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Identification and Biological Evaluation of Secondary Metabolites from the Endolichenic Fungus Aspergillus versicolor by Xiao-Bin Li a ), Yan-Hui Zhou a ), Rong-Xiu Zhu b ), Wen-Qiang Chang a ), Hui-Qing Yuan c ), Wei Gao d ), Lu-Lu Zhang d ), Zun-Tian Zhao d ), and Hong-Xiang Lou* a ) a
) Department of Natural Products Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Science, Shandong University, No. 44 West Wenhua Road, Jinan 250012, P. R. China (phone: þ 86-531-88382012; fax: þ 86-531-88382019; e-mail: [email protected]) b ) School of Chemistry and Chemical Engineering, Shandong University, No. 27 Shanda Nanlu, Jinan 250100, P. R. China c ) Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan 250012, P. R. China d ) College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Jinan 250014, P. R. China
A chemical investigation of the endolichenic fungus Aspergillus versicolor (125a), which was found in the lichen Lobaria quercizans, resulted in the isolation of four novel diphenyl ethers, named diorcinols F – H (1 – 3, resp.) and 3-methoxyviolaceol-II (4), eight new bisabolane sesquiterpenoids, named (¢)-(R)-cyclo-hydroxysydonic acid (5), (¢)-(7S,8R)-8-hydroxysydowic acid (6), (¢)-(7R,10S)-10hydroxysydowic acid (7), (¢)-(7R,10R)-iso-10-hydroxysydowic acid (8), (¢)-12-acetoxy-1-deoxysydonic acid (9), (¢)-12-acetoxysydonic acid (10), (¢)-12-hydroxysydonic acid (11), and (¢)-(R)-11-dehydrosydonic acid (12), two new tris(pyrogallol ethers), named sydowiols D (13) and E (14), and fifteen known compounds, 15 – 29. All of the structures were determined by spectroscopic analyses, and a number of them were further identified through chemical transformations and electronic circular dichroism (ECD) calculations. Preliminary bioassays of these isolates for the determination of their inhibitory activities against the fungus Candida albicans, and their cytotoxicities against the human cancer cell lines PC3, A549, A2780, MDA-MB-231, and HEPG2 were also evaluated.
Introduction. – Among fungi, those that live in close association with other organisms are assumed to be exceptional generators of biologically active compounds [1]. Lichens harbor abundant and diverse fungal communities. Recent studies have demonstrated that endolichenic fungi that live asymptomatically in the thalli of lichens are valuable sources of novel bioactive natural products, such as the antibacterial coniothiepinol A [2], a cyclic pentapeptide with synergistic antifungal activity [3], cytotoxic chromone derivatives [4], perylenequinones [5], ophiobolane sesterterpenes [6], diterpenoid geopyxin B [7], a demethoxyviridin that inhibits Ab42 aggregation [8], and antiviral heptaketides [9]. However, chemical investigations of this group of fungi are rather limited. During our search for new cytotoxic natural products from endolichenic fungi [5], the crude extract of Aspergillus versicolor (125a), which inhabits the lichen Lobaria quercizans, collected in the Laojun Mountain of Yunnan Province in China, exhibited significant cytotoxicity against the human osteosarcoma cancer cell line U2OS with an Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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IC50 value of 21.53 mg/ml. Fractionation of the AcOEt extract of its solid-substrate fermentation culture afforded 14 new compounds, including four diphenyl ethers, 1 – 4, eight bisabolane sesquiterpenoids, 5 – 12, and two pyrogallol ethers, 13 and 14, together with 15 known compounds, 15 – 29. Herein, we describe the isolation, and structure elucidation of the isolated compounds, as well as the evaluation of their antifungal and cytotoxic activities. Results and Discussion. – The AcOEt extract of A. versicolor afforded 29 compounds by using a combination of size-exclusion, normal-phase, and reversedphase chromatography. Structure Elucidation. Diorcinol F (1) was found to have the molecular formula C15H16O5 by a combination of HR-ESI-MS (m/z 277.1072 ([M þ H] þ ; calc. 277.1071)) and 13C-NMR data, accounting for eight degrees of unsaturation. The IR spectrum revealed the presence of OH functions (3355 cm ¢ 1) and aromatic moieties (1662 and 1589 cm ¢ 1). The 1H- and 13C-NMR data, together with the HSQC data (Table 1), exhibited signals of two tertiary Me group (d(H) 2.07, d(C) 20.9, Me¢C(5); d(H) 2.07, d(C) 20.5, Me¢C(5’)), one MeO group (d(H) 3.70, d(C) 60.2, MeO¢C(2)), four aromatic CH groups (d(H) 6.36, d(C) 111.5, CH(4); d(H) 5.97, d(C) 109.3, CH(6); d(H) 6.38, d(C) 111.9, CH(4’); d(H) 6.07, d(C) 110.9, CH(6’)), three exchangeable Hatoms (d(H) 9.16 (2 H); d(H) 8.32 (1 H)), and eight aromatic quaternary C-atoms. Based on these data, compound 1 was deduced to be a dimeric ether consisting of 3,4,5trihydroxytoluene units similar to violaceol I (18) [10], with the exception of the presence of MeO¢C(2) moiety in 1 instead of HO¢C(2) in 18. This conclusion was also supported by the HMBC from MeO¢C(2) to C(2) (d(C) 135.8) (Fig. 1). Accordingly, the structure of compound 1 was unambiguously determined as 3-(3-hydroxy-2methoxy-5-methylphenoxy)-5-methylbenzene-1,2-diol, which is structurally related to the fungal metabolites diorcinols B – E [11]. The trivial name diorcinol F was given to this new compound. Diorcinol G (2) had the molecular formula of C24H30O3 , deduced by the same strategy as for 1, indicating ten degrees of unsaturation. The presence of OH groups and aromatic moieties in 2 was inferred from its IR absorption bands at 3338, 1611, and 1598 cm ¢ 1. The 1D-NMR and HSQC data (Table 1) revealed the presence of six Me, two CH2 , and six aromatic/olefinic CH groups, ten aromatic/olefinic quaternary Catoms, four of which were oxygenated, and two exchangeable H-atoms. These data suggested that 2 was a derivative of diorcinol (16) [12] [13], with two additional prenyl ( ¼ 3-methylbut-2-en-1-yl) groups. One prenyl group at C(2) was determined by the 1 H,1H-COSY correlation H¢C(7) (d(H) 2.97)/H¢C(8) (d(H) 5.06), and the HMBCs from Me(10) (d(H) 1.54) to C(8) (d(C) 122.8) and C(9) (d(C) 130.2), from Me(11) (d(H) 1.48) to C(8) and C(9), and from H¢C(7) to C(1) (d(C) 154.1), C(2) (d(C) 118.6), and C(3) (d(C) 150.5) (Fig. 1). The other prenyl group at C(4) was identified by the 1H,1H-COSY correlation H¢C(12) (d(H) 2.98)/H¢C(13) (d(H) 4.88), and the HMBCs from resonance at Me(15) and Me(16) (d(H) 1.54) to C(13) (d(C) 122.8) and C(14) (d(C) 129.9), and from H¢C(12) to C(3), C(4) (d(C) 123.0), and C(5) (d(C) 135.3) (Fig. 1). Thus, the structure of compound 2 was as depicted. Diorcinol H (3) had the molecular formula C15H14O4 based on HR-ESI-MS (m/z 257.0818 ([M ¢ H] ¢ ; calc. 257.0808)) and 13C-NMR analyses, which revealed nine
a
6.07 (s) 2.07 (s) 2.07 (s) 3.70 (s)
6.38 (s)
5.97 (s)
6.36 (s)
1.54 (s) 1.54 (s)
1.54 (s) 1.48 (s) 2.98 (d, J ¼ 6.0) 4.88 (t, J ¼ 6.0)
2.97 (d, J ¼ 6.0) 5.06 (t, J ¼ 6.0)
5.97 (s) 2.14 (s) 2.12 (s)
6.17 (s)
5.88 (s)
6.54 (s)
d( H )
d(C )
d( H) 150.8 135.8 150.6 111.5 132.7 109.3 144.5 134.4 146.6 111.9 127.5 110.9 20.9 20.5 60.2
2
1 d(C )
23.1 122.8 130.2 25.5 17.5 25.4 122.8 129.9 25.5 17.6
154.1 118.6 150.5 123.0 135.3 114.1 159.2 99.1 158.3 109.2 139.5 106.2 19.2 21.3 2.69 (s) 2.73 (s) 3.87 (s)
6.56 (s)
6.80 (s)
6.60 (s)
d( H )
3 d(C ) 149.1 130.5 147.2 115.0 125.1 116.9 157.3 95.8 155.9 114.2 131.7 115.7 24.0 24.4 60.5 55.9
6.20 (s) 2.06 (s) 2.14 (s) 3.74 (s)
6.20 (s)
5.93 (s)
6.39 (s)
d(C ) 146.8 133.1 148.3 106.6 126.5 107.4 150.6 128.0 150.6 108.2 134.2 108.2 21.1 21.1
d( H)
4
) Recorded at 600 and 150 MHz, respectively. Assignments were based on 1H,1H-COSY, HMQC, and HMBC experiments.
1 2 3 4 5 6 1’ 2’ 3’ 4’ 5’ 6’ 5-Me 5’-Me 2-MeO 3-MeO 7 8 9 10 11 12 13 14 15 16
Position
1.56 (s) 1.56 (s)
3.10 (d, J ¼ 7.2) 5.12 (t, J ¼ 7.2)
6.12 (s) 2.13 (s) 2.15 (s)
6.25 (s)
6.02 (s)
6.15 (s)
6.45 (s)
d(H )
15
22.3 122.8 130.1 25.6 17.6
154.2 117.0 156.3 111.7 136.3 111.5 158.9 101.3 158.4 110.2 139.8 108.5 20.9 21.2
d(C )
Table 1. 1H- and 13C-NMR Data a ) (in (D6 )DMSO) of Compounds 1 – 4 and 15. d in ppm, J in Hz. Atom numbering as indicated in the Formulae.
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Fig. 1. 1H,1H-COSY (— —) and key HMB (H ! C) correlations of compounds 1, 2, 4, 5, 9, 13a, and 14a
degrees of unsaturation. Its IR spectrum revealed the presence of OH groups (3351 cm ¢ 1) and an aromatic system (1612 and 1584 cm ¢ 1). The NMR data of 3 (Table 1) were similar to those of the co-metabolite 3,7-dihydroxy-1,9-dimethyldibenzofuran (20) [14] with the noticeable difference of additional resonances (d(H) 3.87; d(C) 60.5) due to a MeO group at C(2). This finding was supported by the HMBC of MeO¢C(2) to C(2) (d(C) 130.5). Thus, the structure of 3, named diorcinol H, was determined as depicted. 3-Methoxyviolaceol-II (4) was assigned the molecular formula C15H16O5 , which is the same as that of 1, based on a combination of HR-ESI-MS (m/z 277.1072 ([M þ H] þ ; calc. 277.1071)) and 13C-NMR data. The 1H- and 13C-NMR data (Table 1), together with the 2D-NMR data (Fig. 1), of 4 were similar to those of violaceol-II (21) [10]. The HMBC of MeO¢C(3) (d(H) 3.74) with C(3) (d(C) 148.3) evidenced that compound 4 was the 3-O-Me derivative of 21, which is consistent with its molecular formula. (¢)-(R)-Cyclo-hydroxysydonic acid (5) was assigned the molecular formula C15H20O5 by a combination of HR-ESI-MS (m/z 303.1203 ([M þ Na] þ ; calc. 303.1203)) and 13C-NMR data, indicating six degrees of unsaturation. Its IR spectrum revealed absorption bands at 3390 (OH), 1687 (conjugated C¼O group), 1619 and 1605 (aromatic ring) cm ¢ 1. The 1H-NMR data displayed a typical ABX spin system
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assignable to a 1,3,4-trisubstitued benzene ring at d(H) 6.84 (d, J ¼ 7.8, H¢C(3)), 7.35 (s, H¢C(6)), and 7.61 (d, J ¼ 7.8, H¢C(4)). Its 1H- and 13C-NMR, and HSQC data (Table 2) revealed other resonances including those of three tertiary Me (d(H) 1.13, d(C) 29.6, Me(12) and Me(13); d(H) 1.65, d(C) 24.4, Me(14)), three CH2 (d(H) 1.47, d(C) 44.3, CH2(10); d(H) 1.62 – 1.56, d(C) 18.7, CH2(9); d(H) 1.97, d(C) 40.3, CH2(8)) groups, two sp3 oxygenated quaternary C-atoms (d(C) 69.8 (C(7), C(11)), one conjugated COOH (d(C) 167.1, C(15)) group, and six aromatic C-atoms. The 1H,1HCOSY correlations revealed the presence of the spin system CH2(8)¢CH2(9)¢CH2(10) (Fig. 1). Furthermore, the HMBCs (Fig. 1) from H¢C(12) or H¢C(13) (d(H) 1.13) to C(10), C(11), C(12), and C(13), from H¢C(9) to C(7), from H¢C(8) to C(2) (d(C) 121.8) and C(14), and from H¢C(14) to C(2) and C(8) confirmed the presence of an aliphatic side chain at C(2) of the benzene ring. Based on these data, compound 5 was determined as a bisabolane-type sesquiterpenoid similar to hydroxysydonic acid [15], with the exception of the absence of two H-atoms. At present, this compound could be assumed to have the structure of A or B (Fig. 2). However, further comparisons of the 13 C-NMR chemical shifts of C(7) and C(11) in 5 with those of C(1) and C(5) in 1,2dioxepanes confirmed the structure A [16]. The absolute configuration of compound 5 was determined by comparing the experimental and calculated ECD spectra predicted by time-dependent density-functional theory (TDDFT). The absolute configuration of C(7) was assigned as (R) based on the finding that the experimental and calculated ECD spectra of 5 were in good agreement (cf. Fig. 4, a). (¢)-(7S,8R)-8-Hydroxysydowic acid (6), (¢)-(7R,10S)-10-hydroxysydowic acid (7), and (¢)-(7R,10R)-iso-10-hydroxysydowic acid (8) were found to be isomers with the same molecular formula of C15H20O5 , based on a combination of their HR-ESI-MS and 13C-NMR spectra. Their similar 1D-NMR spectra (Table 2) exhibited signals for three Me and two CH2 groups, one CH¢O group, two sp3 quaternary O-bearing Catoms, a 1,3,4-trisubstitued benzene ring containing an oxygenated aromatic C-atom, and one conjugated COOH group. These data suggested that compounds 6 – 8 were hydroxy derivatives of sydowic acid (22) [17]. Compounds 6 and 7 were determined as 8-hydroxy- and 10-hydroxysydowic acids, respectively, by the downfield shifts in the resonances of C(8) (d(C) 69.1) in 6 and of C(10) (d(C) 70.7) in 7. The differences in
Fig. 2. Structures of A, B, and the fragments I, II, III, and IV of compounds 13a and 14a
1.13 (s)
11 12 29.6 24.4 167.1
69.8 29.6
44.3
9.35 (br. s)
1.15 (s) 1.64 (s)
28.0 23.4 170.1
2.04 – 2.00 (m, Ha ), 24.5 1.94 – 1.88 (m, Hb ) 1.94 – 1.88 (m, Ha ), 32.9 1.60 – 1.56 (m, Hb ) 75.4 1.38 (s) 30.4
4.19 (d, J ¼ 6.6)
7.55 (s)
7.22 (d, J ¼ 8.4) 7.55 (d, J ¼ 8.4)
156.6 136.0 125.5 121.4 129.8 119.4 81.3 69.1
d(C)
d(C )
9.11 (br. s)
1.00 (s) 1.55 (s)
1.33 (s)
25.0 31.1 170.7
77.9 26.4
156.9 136.7 7.15 (d, J ¼ 8.4) 124.5 7.57 (d, J ¼ 8.4) 121.7 129.9 7.56 (s) 119.3 77.9 2.30 (dt, J ¼ 14.0, 3.6, Ha ), 27.9 2.14 (td, J ¼ 14.0, 3.6, Hb ) 2.05 (tt, J ¼ 13.2, 3.6, Ha ), 24.1 1.86 – 1.82 (m, Hb ) 3.51 – 3.48 (m) 70.7
d( H )
7 c) d(C)
9.49 (s)
1.19 (s) 1.60 (s)
1.33 (s)
24.9 29.6 170.2
70.7 27.7
155.4 135.5 7.08 (d, J ¼ 7.8) 126.6 7.55 (d, J ¼ 7.8) 121.5 129.7 7.56 (s) 119.3 88.4 2.49 (dt, J ¼ 12.0, 8.4, Ha ), 38.8 2.24 – 2.19 (m, Hb ) 25.5 2.11 – 2.05 (m, Ha ), 1.92 – 1.86 (m, Hb ) 3.89 (t, J ¼ 7.2) 87.0
d( H)
8 c)
2.02 (s)
1.38 – 1.25 (m, Ha ), 1.21 – 1.14 (m, Hb ) 1.38 – 1.25 (m, Ha ), 1.11 – 1.04 (m, Hb ) 1.74 – 1.67 (m) 3.87 (dd, J ¼ 10.0, 5.4, Ha ), 3.79 (dd, J ¼ 10.0, 6.6, Hb ) 0.84 (d, J ¼ 6.6) 1.57 (s)
1.85 – 1.74 (m)
8.06 (d, J ¼ 6.6) 7.52 (d, J ¼ 6.6)
7.52 (d, J ¼ 6.6) 8.06 (d, J ¼ 6.6)
d( H )
9 c)
171.5 21.1
16.9 30.3 171.5
32.5 69.5
33.6
21.3
154.0 125.1 130.3 127.9 130.3 125.1 74.9 44.3
d(C )
a ) Recorded at 600 and 150 MHz, respectively. Assignments were based on 1H,1H-COSY, HMQC, HMBC and NOESY experiments. b ) Data were recorded in ( D6 )acetone. c ) Data were recorded in CDCl3 .
1.13 (s) 1.65 (s)
1.47 (t, J ¼ 7.8)
10
13 14 15 1-OH 12-AcO 12-AcO
1.62 – 1.56 (m)
9
18.7
152.5 121.8 6.84 (d, J ¼ 7.8) 108.4 7.61 (d, J ¼ 7.8) 125.6 148.7 7.35 (s) 109.7 69.8 1.97 (t, J ¼ 7.8) 40.3
d( H)
d(H )
d(C )
6 c)
5 b)
1 2 3 4 5 6 7 8
Position
Table 2. 1H- and 13C-NMR Data of Compounds 5 – 9 a ). d in ppm, J in Hz.
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the 13C chemical shifts in the heterocyclic rings of 7 and 8 were consistent with those of linalool oxides [18] [19]. The relative configurations of these three compounds were determined by their NOESY correlations (Fig. 3). The NOEs H¢C(8)/Hb¢C(10), Hb¢C(10)/Me(13), and Me(14)/Me(12) in 6 indicated that H¢C(8) and Me(14) were on different faces. The correlations H¢C(10)/Ha¢C(9), Ha¢C(9)/Ha¢C(8), Me(14)/ Hb¢C(8), and Hb¢C(8)/Hb¢C(9) in 7 evidenced that H¢C(10) and Me(14) were on different faces. Similarly, the NOEs Me(14)/Ha¢C(9) and Hb¢C(9)/H¢C(10) in 8 indicated that Me(14) and H¢C(10) were on different faces. The absolute configurations of compounds 6 – 8 were determined by comparing the experimental and calculated ECD spectra predicted by TDDFT (Fig. 4, b – 4, d). The experimental and calculated ECD spectra of 6 were in good agreement. The calculated ECD spectra of 7 and of 8 were nearly mirror images of their experimental ECD spectra, respectively. (¢)-12-Acetoxy-1-deoxysydonic acid (9) was found to have the molecular formula C17H24O5 based on its HR-ESI-MS (m/z 331.1520 ([M þ Na] þ ; calc. 331.1516)) and 13 C-NMR data, which revealed six degrees of unsaturation. The 1D-NMR data (Table 2) resembled those of 12-acetoxysydonic acid [20] with the exception of the A2B2 spin system (d(H) 7.52 (d, J ¼ 6.6, H¢C(2) and H¢C(6)) and 8.06 (d, J ¼ 6.6, H¢C(3) and H¢C(5))) in 9 rather than the ABX spin system in 12-acetoxysydonic acid, indicating the absence of a phenolic OH group in 9. The planar structure of 9 was confirmed by detailed analysis of its 2D-NMR data (Fig. 1).
Fig. 3. Selected NOESY correlations (H $ H) of compounds 6 – 8
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Fig. 4. Experimental and calculated ECD spectra of compound 5 (a), 6 (b), experimental ECD spectrum for 7 and calculated ECD spectrum for (7S,10R)-7 (c), and experimental ECD spectrum for compound 8 and calculated ECD spectrum for (7S,10S)-8 (d)
Compounds 10, 11, and 12 were found to be the 7-epimers of (þ)-(7S,11S)-12acetoxysydonic acid [20], (þ)-(7S,11S)-12-hydroxysydonic acid [21], and (þ)-(S)11-dehydrosydonic acid [20], respectively, based on comparisons of their NMR spectra, MS data, and optical rotation values with those in the literature. Because there was only one stereogenic C-atom, the absolute configuration at C(7) in 12 was assigned as (R) by comparing its negative optical rotation value ([a] 25 D ¼ ¢ 30.0 (c ¼ 0.1, MeOH)) with the positive optical rotation value of (þ)-(S)-11-dehydrosydonic acid ([a] 23 D ¼ þ 10.8 (c ¼ 0.11, MeOH)) [20]. From biogenetic considerations (Scheme), compounds 9 – 11 can be assumed to have the same absolute configuration, (7R), as that of 12. The molecular formulae of sydowiols D and E (13 and 14, resp.) were both determined as C21H20O7 based on their HR-ESI-MS data (m/z 383.1128 ([M ¢ H] ¢ ; calc. 383.1125)). Their 1H- and 13C-NMR spectroscopic data, which were similar to those of the co-metabolites sydowiols A and B (24 and 25, resp.) [22], indicated that both 13 and 14 consist of three ether-linked 3,4,5-trihydroxytoluene moieties. In our study, although compounds 13 and 14 could be separated by HPLC, these were
Scheme 1. Possible Biosynthetic Pathways to Compounds 1 – 15
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observed to be isomerized after standing in MeOH or MeCN. The methylation of a mixture 13/14 (MeI/K2CO3 ) afforded pentamethyl ethers 13a (C26H30O7 ) and 14a (C26H30O7 ), indicating the presence of five OH groups in 13 and 14. In the HMBC spectrum of 13a (Fig. 1), the correlations from H¢C(4) (d(H) 6.49) to C(2) (d(C) 137.8), attached to a MeO group (d(H) 3.89, d(C) 61.2), and C(3) (d(C) 153.4) attached to a MeO group (d(H) 3.87, d(C) 56.2), from Me¢C(5) (d(H) 2.25) to C(4) (d(C) 108.2), C(5) (d(C) 133.6), and C(6) (d(C) 112.6), and from H¢C(6) (d(H) 6.37) to O-bearing C(1) (d(C) 150.5) and C(2) indicated the partial structure I (Fig. 2). The HMBCs from H¢C(4’) (d(H) 6.08) to C(2’) (d(C) 137.9), attached to a MeO group (d(H) 3.99, d(C) 61.2), and O-bearing C(3’) (d(C) 152.6), from Me¢C(5’) (d(H) 2.07) to C(4’) (d(C) 110.1), C(5’) (d(C) 133.2), and C(6’) (d(C) 113.3), and from H¢C(6’) (d(H) 6.29) to oxygenated C(1’) (d(C) 150.3) and C(2’) indicated moiety II, whereas moiety III was suggested by the HMBCs from two H-atoms with signals at d(H) 6.46 (H¢C(4’’) and H¢C(6’’)) to C-atoms with signals at d(C) 153.1 (C(1’’) and C(3’’)), attached to MeO groups (d(H) 3.77, d(C) 56.3), and oxygenated C(2’’) (d(C) 130.3), and from Me(5’’) (d(H) 2.38) to C(4’’) (d(C) 106.3), C(5’’) (d(C) 135.5), and C(6’’) (d(C) 106.3), in addition to symmetry considerations. Due to the presence of a single MeO group, fragment II had obviously to be placed between fragments I and III. The structure of 14a was deduced by the same strategy that was used for the deduction of 13a. Three moieties, I, III, and IV (Fig. 2), were indicated by the detailed analysis of the HMBC data of 14a (Fig. 1), and III was connected to IV at C(1’) (d(C) 152.4) via an Oatom based on the HMBC between H¢C(6’) (d(H) 6.03) and C(2’’) (d(C) 130.3). Thus, 14a was unambiguously determined, and sydowiols D and E (13 and 14, resp.) were identified as shown. The assignments for the 1H- and 13C-NMR data of 13a and 14a are compiled in Table 3. Table 3. 1H- and 13C-NMR Data (in CDCl3 ) of Compounds 13a and 14a a ). d in ppm, J in Hz. Position
13a d( H )
1 2 3 4 5 6 2-MeO 3-MeO 5-Me 1’ 2’ 3’ 4’ 5’
6.49 (s) 6.37 (s) 3.89 (s) 3.87 (s) 2.25 (s)
6.08 (s)
14a d(C ) 150.5 137.8 153.4 108.2 133.6 112.6 61.2 56.2 21.7 150.3 137.9 152.6 110.1 133.2
d( H)
6.36 (s) 6.30 (s) 3.99 (s) 3.84 (s) 2.22 (s)
6.41 (s)
Position
13a d( H)
d(C )
d( H )
d(C )
6’ 2’-MeO 3’-MeO 5’-Me 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 1’’-MeO 3’’-MeO 5’’-Me
6.29 (s) 3.99 (s)
113.3 61.2
6.03 (s)
107.3
2.07 (s)
21.5 153.1 130.3 153.1 106.3 135.5 106.3 56.3 56.3 22.2
3.74 (s) 2.22 (s)
56.3 22.0 153.2 130.3 153.2 106.8 135.3 106.8 56.5 56.5 22.1
d(C ) 152.2 135.9 153.0 106.4 132.7 108.6 61.1 56.2 21.9 152.4 135.6 153.3 106.6 134.9
6.46 (s) 6.46 (s) 3.77 (s) 3.77 (s) 2.38 (s)
14a
6.41 (s) 6.41 (s) 3.69 (s) 3.69 (s) 2.33 (s)
a ) Recorded at 600 and 150 MHz, respectively. Assignments were based on HMQC and HMBC experiments.
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Diorcinol I (15) had the molecular formula of C19H22O3 , as established by a combination of HR-ESI-MS (m/z 299.1640 ([M þ H] þ ; calc. 299.1642)) and 1D-NMR data, indicating nine degrees of unsaturation. The UV, IR, and 1D-NMR data of 15 were similar to those of diorcinol G (2). Analyses of its 2D-NMR data revealed that the prenyl group at C(4) in 2 was absent in 15, and this finding was supported by the HMBCs from H¢C(4) (d(H) 6.45) to C(2) (d(C) 117.0), C(3) (d(C) 156.3), C(5) (d(C) 136.3), and C(6) (d(C) 111.5). This is the first report on the structure of 15, which was named diorcinol I. The known compounds were identified as diorcinol (16) [13], diorcinol D (17) [11], violaceol I (18) [23], cordyol C (19) [24], 3,7-dihydroxy-1,9-dimethyldibenzofuran (20) [14], violaceol II (21) [23], sydowic acid (22) [17], sydonic acid (23) [25], sydowiol A (24) [22], sydowiol B (25) [22], aversin (26) [26], 6,8-di-O-methylnidurufin (27) [27], 4hydroxybenzaldehyde (28) [28], and 3-formyl-1H-indole (29) [29] by comparing their spectroscopic data (MS, [a]D, UV, IR, and 1D-NMR) with those reported previously. Phenyl ether derivatives which turned out to be the major components in this study were previously obtained from cultured lichen mycobionts [10] [14] [30], lichens [31] [32], sponges [33] [34], and the fungi Aspergillus sp. [11 – 13] [21 – 23] [35 – 40], Penicillium sp. [20] [41], Hypocrea citrina [42], Emericella falconensis [43], Emericella violacea [44], and Cordyceps sp. [24]. To date, only one report appeared on the isolation of phenyl ethers from an endolichenic fungus [40]. It is noteworthy that phenyl ethers from natural lichens generally have carboxy groups, such as micareic acid, while phenyl ethers from the endolichenic fungus [40] and the cultured mycobionts [10] [14] [30] have no such group. In addition, this is the first report on the presence of bisabolane sesquiterpenes in an endolichenic fungus. Plausible biosynthetic routes to the new phenyl ethers and bisabolane sesquiterpenoids are outlined in the Scheme. The phenyl ethers are closely related to orsellinic acid and gallic acid, which are derived from the acetate-malonate pathway [45] and shikimic acid pathway [46], respectively. A series of dehydration, methylation, cyclization, oxidation reactions, and the addition of dimethylallyl diphosphate (DMAPP) unit to 16 lead to the formation of phenyl ethers 1 – 4 and 13 – 15. The bisabolane nucleus is built up via the mevalonic acid pathway [21]. Subsequently, 5 – 12 are formed through a series of oxidation, reduction, cyclization reactions, and esterification of the bisabolane nucleus. Biological Activities. All of the isolated compounds were evaluated for their cytotoxic activities against the PC3 (human prostate adenocarcinoma), A549 (human lung adenocarcinoma), A2780 (human ovarian carcinoma), MDA-MB-231 (human metastatic breast adenocarcinoma), and HEPG2 (human hepatocellular carcinoma) cancer cell lines by using the MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) assay [47]. As shown in Table 4, the cytotoxic activity displayed by A. versicolor (125a) was attributed to the presence of phenyl ethers. Among these, compounds 2 and 15 exhibited moderate cytotoxicities against all of the tested human cancer cell lines with IC50 values ranging from 19.0 to 36.3 mm. Compounds 3, 20, and 17 showed weak cytotoxicities against one to three of the tested human cancer cell lines. The results support the conclusion that the prenyl group (in 2 and 15) enhances cytotoxic activity, whereas an additional MeO group (in 3) reduces the activity. No cytotoxic activity was observed with the bisabolane sesquiterpenoids. However, it has
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Table 4. Cytotoxic and Antifungal Activities of Compounds a ) Compound
IC50 [mm] b ) c
3 2 15 20 17 18/21 19 27 Cisplatin Fluconazole
MIC (mg/ml) d
e
g
PC3 )
A549 )
A2780 )
MDA-MB231 f )
HEPG2 )
Candida albicans
> 50 24.0 0.5 24.7 1.4 39.4 1.7 31.6 0.8 > 50 > 50 31.9 2.3 18.0 1.5 NT
> 50 23.9 1.8 26.2 3.6 > 50 > 50 > 50 > 50 > 50 19.9 1.7 NT
> 50 31.0 0.6 36.3 2.1 > 50 > 50 > 50 > 50 33.7 2.5 14.9 2.3 NT
43.2 2.7 19.0 1.1 23.2 2.1 44.9 0.9 > 50 > 50 > 50 28.3 1.8 18.0 1.6 NT
> 50 24.3 0.9 27.3 1.7 45.2 1.2 29.8 1.0 > 50 > 50 > 50 14.3 1.5 NT
> 64 > 64 32 64 8 8 8 > 64 NT h ) 2
a
) Compounds which are not listed were inactive in all assays. Cisplatin and DMSO were used as the positive and negative controls, respectively, for the cytotoxicity assays; fluconazole and DMSO were used as the positive and negative controls, respectively, for the antifungal activity assay. b ) The IC50 values are means standard deviation of three independent replicates. c ) Human prostate adenocarcinoma. d ) Human lung adenocarcinoma. e ) Human ovarian carcinoma. f ) Human metastatic breast adenocarcinoma. g ) Human hepatocellular carcinoma. h ) NT, Not tested.
been reported that the condensation between phenolic bisabolane-type sesquiterpenoids and diphenyl ethers may increase their cytotoxicity [20]. The inhibitory activities of the isolated metabolites were also assessed against the fungus Candida albicans by using the microdilution method [48]. The known compounds 17, the mixture 18/21, and 19 exhibited significant activities with an MIC value of 8 mg/ml. Compounds 15 and 20 exhibited weak antifungal activities with MIC values of 32 and 64 mg/ml, respectively. All of the new compounds were inactive displaying MIC values greater than 64 mg/ml. Conclusions. – We reported 14 new compounds, including four diphenyl ethers, 1 – 4, eight bisabolane sesquiterpenoids, 5 – 12, and two pyrogallol ethers, 13 and 14, together with 15 known compounds, 15 – 29, isolated from the endolichenic fungus A. versicolor (125a). Phenyl ethers were confirmed as the major bioactive components in the extract of A. versicolor (125a) by evaluation of cytotoxic and antifungal activities. In addition, plausible biogenetic pathways of the new phenyl ethers and bisabolane sesquiterpenoids are also proposed. This work was supported by the National Natural Science Foundation of China (No. 81273383). We kindly acknowledge Mr. Shu-Qi Wang and Mr. Bin Ma for recording the NMR spectra, and Mrs. Yan-Hui Gao for the HR-ESI-MS data. Experimental Part General. TLC: Pre-coated SiO2 GF254 plates (Qingdao Marine Chemical Industry), visualization by using UV (254 nm) light or by spraying with 10% H2SO4/EtOH, followed by heating. Column chromatography (CC): silica gel (SiO2 , 200 – 300 mesh; Qingdao Marine Chemical Industry), octadecyl
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silica gel (ODS, 40 – 63 mm; Fuji Silysia Chemical, Ltd.), and Sephadex LH-20 gel (25 – 100 mm; Pharmacia Biotech). MPLC: Lisure EZ Purifier apparatus equipped with a UV/VIS dual-wavelength detector (210 and 254 nm; Lisure Science Corporation) and an ODS column (30 130 mm). Semi-prep. HPLC: Agilent 1100-G1310A isopump equipped with a G1322A degasser, a G1314A VWD detector (210 nm), and a ZORBAX SB-C18 column (9.4 250 mm, 5 mm). Optical rotations: Perkin-Elmer 241 MC polarimeter. UV Spectra: Shimadzu UV-2450 spectrophotometer; lmax (log e) in nm. CD Spectra: Chirascan spectropolarimeter; l (De) in nm. IR Spectra: Nicolet iN 10 Micro FTIR spectrometer; ˜n in cm ¢ 1. 1H- and 13C-NMR Spectra: Bruker Avance-DRX-600 spectrometer; at 600 (1H) and 150 MHz (13C); d in ppm rel. to Me4Si as internal standard, J in Hz; 2D spectra recorded with standard pulse programs and acquisition parameters. MS: API-4000 triple-stage quadrupole instrument for electrospray ionization (ESI) and Finnigan LC-QDECA mass spectrometer for HR-ESI; in m/z. Fungal Material. The endolichenic fungus A. versicolor (125a) was isolated from the lichen Lobaria quercizans, which was collected from Laojun Mountain, Yunnan Province, P. R. China. It was identified by Xin Zhang (Shandong Provincial Key Laboratory of Microbial Engineering, Qilu University of Technology, P. R. China) based on its nuclear ITS rDNA sequences (GenBank: GU227343). The strain, which was assigned the Accession No. 125a, was deposited with the lichen laboratory in the College of Life Sciences, Shandong Normal University, Jinan. The fungal strain was cultured in two 500-ml Erlenmeyer flasks, each of which contained 100 ml of potato dextrose broth (PDB), at 258 on a rotary shaker (110 rpm) for 8 d to prepare the seed culture. The fermentation was conducted in 20 Erlenmeyer flasks (500 ml), each of which contained 80 g of rice. Dist. H2O (120 ml) was added to each flask, and the contents were soaked overnight before autoclaving at 1208 for 30 min. After cooling to r.t., each flask was inoculated with 10 ml of the spore inoculum and incubated under static conditions at r.t. for 60 d. Extraction and Isolation. The culture medium containing the mycelium was cut into small pieces and extracted using AcOEt (3 8 l). The org. solvent was evaporated under reduced pressure to yield the crude extract (10.9 g), which exhibited cytotoxicity against the human U2OS cell line (IC50 50.35 mg/ml). The AcOEt extract was separated into ten fractions, Frs. A – J, on a SiO2 column (CH2Cl2/MeOH from 100 : 0 to 0 : 100). Fr. B (49.5 mg) was subjected to gel permeation chromatography (Sephadex LH-20; CH2Cl2/MeOH 1 : 1) to afford four subfractions, Frs. B1 – B4 . Fr. B3 (37.5 mg) was purified by HPLC (83% MeOH/H2O, 1.5 ml/min) to yield 2 (tR 46.0 min; 5.0 mg) and 15 (tR 18.0 min; 21.4 mg). Fr. D (1.3 g) was subjected to CC (Sephadex LH-20; same solvent system as described above) to afford four subfractions, Frs. D1 – D4 . Fr. D2 (213.2 mg) was purified by HPLC (71% MeOH/H2O; 1.8 ml/ min) to yield 26 (tR 38.0 min; 7.7 mg) and 27 (tR 41.0 min; 6.0 mg). Fr. D3 (944.0 mg) was separated by MPLC (ODS, MeOH/H2O from 50 : 50 to 100 : 0) to yield six subfractions, Frs. D3A – D3F . Fr. D3A (328.9 mg) was further purified by HPLC (60% MeOH/H2O; 1.8 ml/min) to afford 28 (tR 7.0 min; 23.5 mg), 29 (tR 9.0 min; 5.5 mg), 4 (tR 14.0 min; 5.2 mg), 1 (tR 18.0 min; 4.3 mg), and 3 (tR 26.3 min; 2.1 mg). Further separation of Fr. D3C (55.3 mg) by HPLC (71% MeOH/H2O, 1.8 ml/min) yielded 22 (tR 25.9 min; 18.2 mg). Fr. D3D (126.6 mg) was purified by HPLC (76% MeOH/H2O; 1.8 ml/min) to furnish 17 (tR 24.0 min; 90.7 mg). Compounds 20 (tR 13.8 min; 8.8 mg) and 16 (tR 17.3 min; 7.9 mg) were isolated from Fr. D4 (20.5 mg) by using HPLC (65% MeOH/H2O; 1.8 ml/min). Fr. F (4.5 g) was separated by CC (Sephadex LH-20; CH2Cl2/MeOH 1 : 1) to afford five subfractions, Frs. F1 – F5 . Of these, Fr. F3 (747.0 mg) was subjected to HPLC (60% aq. MeOH containing 0.2% CF3COOH) to yield 5 (tR 30.0 min; 0.5 mg), 9 (tR 27.0 min; 1.1 mg), 10 (tR 22.0 min; 67.1 mg), 11 (tR 24.0 min; 4.3 mg), and 12 (tR 37.0 min; 7.5 mg). The separation of the residue of Fr. F3 , which was performed using a procedure similar to that used for the separation of Fr. D3 , afforded 6 (HPLC; 28% MeCN/H2O (0.2% CF3COOH); 1.5 ml/min; tR 39.0 min, 1.1 mg), 7 (HPLC; 25% MeCN/H2O (0.2% CF3COOH); 1.8 ml/min; tR 24.0 min, 0.2 mg), 8 (HPLC; 28% MeCN/H2O (0.2% CF3COOH); 1.5 ml/ min; tR 31.0 min, 0.6 mg), and 23 (HPLC; 50% MeCN/H2O (0.2% CF3COOH); 1.8 ml/min; tR 20.5 min, 2.8 mg). Fr. F4 (3.4 g) was purified by MPLC (ODS; 50% MeOH/H2O) to yield four subfractions, Frs. F4A – F4D. Fr. F4A (2.7 g) was further purified by HPLC (47% MeOH/H2O; 1.8 ml/min) to yield 18 (tR 30.5 min; 652.8 mg) and 21 (tR 21.0 min; 572.2 mg) as the major compounds. Compounds 18 and 21 were separated as single compounds but isomerized to give a mixture after standing in MeOH, as reported in [10]. Fr. F4B (219.8 mg) was purified by HPLC (55% MeOH/H2O; 1.8 ml/min) to yield 19 (tR 21.0 min;
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24.3 mg), whereas the separation of Fr. F4C (162.4 mg) by HPLC (40% MeCN/H2O; 1.8 ml/min) afforded 13 (tR 23.6 min; 19.5 mg), 14 (tR 18.9 min; 14.5 mg), 24 (tR 27.8 min; 13.9 mg), and 25 (tR 21.4 min; 20.9 mg). Interconversions of 13 and 14, and of 24 and 25 were observed similarly to those of 18 and 21. Diorcinol F ( ¼ 3-( 3-Hydroxy-2-methoxy-5-methylphenoxy)-5-methylbenzene-1,2-diol; 1). Red amorphous solid. UV (MeOH): 278 (5.33), 227 (6.31). IR: 3355, 2933, 1662, 1589, 1510, 1459, 1342, 1320, 1229, 1193, 1138, 1076, 1022, 990, 826. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 277.1072 ([M þ H] þ , C15H17O þ5 ; calc. 277.1071). Diorcinol G ( ¼ 3-(3-Hydroxy-5-methylphenoxy)-5-methyl-2,6-bis(3-methylbut-2-en-1-yl)phenol; 2). Brown oil. UV (MeOH): 281 (3.65). IR: 3338, 2965, 2922, 2856, 1611, 1598, 1449, 1321, 1154, 1136, 1075, 1054, 1023, 991, 835. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 367.2267 ([M þ H] þ , C24H31O þ3 ; calc. 367.2268). Diorcinol H ( ¼ 4-Methoxy-1,9-dimethyldibenzo[b,d]furan-3,7-diol; 3). Brown amorphous solid. UV (MeOH): 231 (4.10). IR: 3351, 2971, 2927, 2851, 1612, 1584, 1519, 1450, 1407, 1383, 1269, 1241, 1200, 1149, 1135, 1079, 1000, 838. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 257.0818 ([M ¢ H] ¢ , C15H13O ¢4 ; calc. 257.0808). 3-Methoxyviolaceol-II ( ¼ 2-(2-Hydroxy-3-methoxy-5-methylphenoxy)-5-methylbenzene-1,3-diol; 4). Red amorphous solid. UV (MeOH): 278 (3.78), 228 (4.68). IR: 3278, 2940, 2861, 1604, 1515, 1463, 1427, 1361, 1319, 1206, 1143, 1083, 1063, 1022, 984, 820. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 277.1072 ([M þ H] þ , C15H17O þ5 ; calc. 277.1071). ( ¢ )-(R)-Cyclo-hydroxysydonic Acid ( ¼ (3R)-3-(4-Hydroxy-4-methylpentyl)-3-methyl-3H-1,2-benzodioxole-6-carboxylic Acid; 5). Colorless amorphous solid. [a] 25 D ¼ ¢ 40.0 (c ¼ 0.1, MeOH). UV (MeOH): 294 (3.83), 258 (3.85). CD (MeOH): 297 ( ¢ 1.83), 262 ( ¢ 1.81), 227 ( ¢ 2.30). IR: 3390, 3077, 2968, 1687, 1619, 1605, 1497, 1452, 1381, 1263, 1184, 1118, 1022, 941, 905, 829, 766. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 303.1203 ([M þ Na] þ , C15H20NaO þ5 ; calc. 303.1203). ( ¢ )-(7S,8R)-8-Hydroxysydowic Acid ( ¼ 3-Hydroxy-4-[(2S,3R)-3-hydroxy-2,6,6-trimethyltetrahydro-2H-pyran-2-yl]benzoic Acid; 6). Colorless amorphous solid. [a] 25 D ¼ ¢ 30.0 (c ¼ 0.1, MeOH). UV (MeOH): 281 (3.73), 228 (4.48). CD (MeOH): 298 ( þ 3.59), 250 ( ¢ 4.08). IR: 3242, 2972, 2937, 1699, 1572, 1511, 1438, 1370, 1261, 1220, 1130, 1103, 1065, 1046, 984, 956, 892, 771, 747. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 279.1232 ([M ¢ H] ¢ , C15H19O ¢5 ; calc. 279.1227). ( ¢ )-(7R,10S)-10-Hydroxysydowic Acid ( ¼ 3-Hydroxy-4-[(2R,5S)-5-hydroxy-2,6,6-trimethyltetrahydro-2H-pyran-2-yl]benzoic Acid; 7). Colorless amorphous solid. [a] 25 D ¼ ¢ 20.0 (c ¼ 0.1, MeOH). UV (MeOH): 278 (4.47), 228 (5.26). CD (MeCN): 255 ( þ 2.69), 240 ( þ 3.33), 225 ( þ 2.53), 218 ( ¢ 1.75). IR: 3263, 2972, 1694, 1573, 1511, 1436, 1378, 1267, 1221, 1180, 1108, 1068, 1038, 963, 833, 771, 749. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 279.1232 ([M ¢ H] ¢ , C15H19O ¢5 ; calc. 279.1227). ( ¢ )-(7R,10R)-Iso-10-hydroxysydowic Acid ( ¼ 3-Hydroxy-4-[(2R,5R)-5-(2-hydroxypropan-2-yl)-2methyltetrahydrofuran-2-yl]benzoic Acid; 8). Colorless amorphous solid. [a] 25 D ¼ ¢ 30.0 (c ¼ 0.1, MeOH). UV (MeOH): 283 (3.69), 228 (4.40). CD (MeCN): 297 ( ¢ 0.82), 248 ( ¢ 2.23). IR: 3241, 2974, 2930, 1694, 1574, 1510, 1417, 1375, 1289, 1212, 1182, 1095, 1062, 1022, 948, 892, 772, 749. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 303.1205 ([M þ Na] þ , C15H20NaO þ5 ; calc. 303.1203). ( ¢ )-12-Acetoxy-1-deoxysydonic Acid ( ¼ 4-[(2R,6S)-7-(Acetyloxy)-2-hydroxy-6-methylheptan-2yl]benzoic Acid; 9). Colorless oil. [a] 25 D ¼ ¢ 60.0 (c ¼ 0.1, MeOH). UV (MeOH): 227 (3.87). CD (MeCN): 245 ( ¢ 3.21), 233 ( ¢ 5.13). IR: 3436, 2941, 1715, 1611, 1461, 1409, 1392, 1373, 1249, 1183, 1124, 1036, 1019, 862, 780, 709. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 331.1520 ([M þ Na] þ , C17H24NaO þ5 ; calc. 331.1516). Sydowiol D ( ¼ 3-[3-(2,6-Dihydroxy-4-methylphenoxy)-2-hydroxy-5-methylphenoxy]-5-methylbenzene-1,2-diol; 13)/Sydowiol E ( ¼ 3-[2-(2,6-Dihydroxy-4-methylphenoxy)-6-hydroxy-4-methylphenoxy]5-methylbenzene-1,2-diol; 14). Red amorphous solid. UV (MeOH): 277 (3.42). IR: 3388, 2923, 1703, 1598, 1509, 1461, 1312, 1189, 1065, 827. HR-ESI-MS: 383.1128 ([M ¢ H] ¢ , C21H19O ¢7 ; calc. 383.1125). Methylation of 13 and 14. MeI (0.2 ml) and K2CO3 (62.3 mg) were added to a mixture 13/14 (5.8 mg) in MeCN (7 ml), and the suspension was stirred at 408 for 50 h. At this time, TLC indicated the disappearance of 13 and 14. The mixture was cooled to r.t., and the reaction was quenched with 2m HCl soln. (22 ml). The mixture was diluted with H2O and extracted with CH2Cl2 (30 ml 3). The org. layers were combined, washed with H2O, saturated with NaCl, dried (MgSO4 ), filtered, and concentrated in
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vacuo. The residue was purified by HPLC (75% MeCN/H2O; 1.8 ml/min) to yield 13a (0.6 mg) and 14a (1.3 mg). 2,3,2’,1’’,3’’-Penta-O-methylsydowiol D ( ¼ 1-(2,3-Dimethoxy-5-methylphenoxy)-3-(2,6-dimethoxy-4methylphenoxy)-2-methoxy-5-methylbenzene; 13a). Colorless amorphous solid. UV (MeOH): 270 (3.53), 206 (5.26). IR: 2937, 2836, 1596, 1578, 1503, 1462, 1415, 1323, 1223, 1130, 1104, 1042, 1005, 967, 816, 775. 1H- and 13C-NMR: Table 3. HR-ESI-MS: 455.2068 ([M þ H] þ , C26H31O þ7 ; calc. 455.2064). 2,3,1’,1’’,3’’-Penta-O-methylsydowiol E ( ¼ 2-(2,3-Dimethoxy-5-methylphenoxy)-1-(2,6-dimethoxy-4methylphenoxy)-3-methoxy-5-methylbenzene; 14a). Colorless amorphous solid. UV (MeOH): 269 (3.01), 207 (4.92). IR: 2917, 2850, 1596, 1504, 1465, 1415, 1324, 1222, 1129, 1094, 1005, 811, 717. 1H- and 13 C-NMR: Table 3. HR-ESI-MS: 477.1879 ([M þ Na] þ , C26H30NaO þ7 ; calc. 477.1884). Diorcinol I ( ¼ 3-(3-Hydroxy-5-methylphenoxy)-5-methyl-2-(3-methylbut-2-en-1-yl)phenol; 15). Brown oil. UV (MeOH): 281 (3.30). IR: 3324, 2965, 2920, 2857, 1587, 1496, 1453, 1420, 1325, 1211, 1143, 1054, 1023, 989, 834. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 299.1640 ([M þ H] þ , C19H23O þ3 ; calc. 299.1642). Biological Assays. Cytotoxicities of the compounds against the PC3, A549, A2780, MDA-MB-231, and HEPG2 cell lines were evaluated using the MTT assay [45], with cisplatin as positive control. Growth inhibitory activity against the fungus Candida albicans was assessed by the microdilution method [46], with fluconazole as positive control. Candida albicans was obtained from Affiliated Qianfoshan Hospital of Shandong University, P. R. China. Theory and Calculation Details. The calculations were performed with the Gaussian 09 program package [49]. Conformational searches were performed by MD simulations based on the COMPASS force field [50] and by scanning the potential energy surface (PES) on the main chain dihedral angles using the semiempirical AM1. All ground-state geometries were optimized at the B3LYP/6-31G* level at 298 K, followed by calculations of their harmonic frequency analysis to confirm these minima and then calculations of r.t. free energies. Electronic excitation energies and rotational strengths in gas phase and in MeOH/MeCN were calculated using TDDFT [51] at the same level in velocity formalism for the first 60 states. The solvent effect of MeOH/MeCN has been modeled by a conductor-like screening model for real solvents (COSMO) [52] [53]. The rotatory strengths were summed and energetically weighted following the Boltzmann statistics, and the ECD curves were simulated by using the Gaussian function [54]: De ðEÞ ¼
X 2 1 1 pffiffiffi DEi Ri e¢½ðE¢DEi Þ=s¤ ¢39 2:296 10 s p i
where s is half the band width at 1/e height, and DEi and Ri are the excitation energies and rotatory strengths for transition i, resp. A s value of 0.4 eV was applied for calculations.
REFERENCES [1] M. M. Wagenaar, D. M. Gibson, J. Clardy, Org. Lett. 2002, 4, 671. [2] Y.-C. Wang, S.-B. Niu, S.-C. Liu, L.-D. Guo, Y.-S. Che, Org. Lett. 2010, 12, 5081. [3] W. Wu, H.-Q. Dai, L. Bao, B. Ren, J.-C. Lu, Y.-M. Luo, L.-D. Guo, L.-X. Zhang, H.-W. Liu, J. Nat. Prod. 2011, 74, 1303. [4] F. Zhang, L. Li, S.-B. Niu, Y.-K. Si, L.-D. Guo, X.-J. Jiang, Y.-S. Che, J. Nat. Prod. 2012, 75, 230. [5] G. Li, H.-Y. Wang, R.-X. Zhu, L.-M. Sun, L.-N. Wang, M. Li, Y.-Y. Li, Y.-Q. Liu, Z.-T. Zhao, H.-X. Lou, J. Nat. Prod. 2012, 75, 142. [6] Q.-X. Wang, L. Bao, X.-L. Yang, D.-L. Liu, H. Guo, H.-Q. Dai, F.-H. Song, L.-X. Zhang, L.-D. Guo, S.-J. Li, H.-W. Liu, Fitoterapia 2013, 90, 220. [7] E. M. K. Wijeratne, B. P. Bashyal, M. X. Liu, D. D. Rocha, G. M. K. B. Gunaherath, J. M. UÏRen, M. K. Gunatilaka, A. E. Arnold, L. Whitesell, A. A. L. Gunatilaka, J. Nat. Prod. 2012, 75, 361. [8] Q.-C. Zheng, G.-D. Chen, M.-Z. Kong, G.-Q. Li, J.-Y. Cui, X.-X. Li, Z.-Y. Wu, L.-D. Guo, Y.-Z. Cen, Y.-Z. Zheng, H. Gao, Steroids 2013, 78, 896.
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[9] J.-W. He, G.-D. Chen, H. Gao, F. Yang, X.-X. Li, T. Peng, L.-D. Guo, X.-S. Yao, Fitoterapia 2012, 83, 1087. [10] Y. Takenaka, T. Tanahashi, N. Nagakura, N. Hamada, Chem. Pharm. Bull. 2003, 51, 794. [11] H.-Q. Gao, L.-N. Zhou, S.-X. Cai, G.-J. Zhang, T.-J. Zhu, Q.-Q. Gu, D.-H. Li, J. Antibiot. 2013, 66, 539. [12] J. A. Ballantine, C. H. Hassall, B. D. Jones, Phytochemistry 1968, 7, 1529. [13] A. N. Yurchenko, O. F. Smetanina, A. I. Kalinovsky, M. V. Pivkin, P. S. Dmitrenok, T. A. Kuznetsova, Russ. Chem. Bull., Int. Ed. 2010, 59, 852. [14] T. Tanahashi, Y. Takenaka, N. Nagakura, N. Hamada, Phytochemistry 2001, 58, 1129. [15] T. Hamasaki, K. Nagayama, Y. Hatsuda, Agric. Biol. Chem. 1978, 42, 37. [16] H. Kropf, H. Von Wallis, J. Chem. Res., Synop. 1983, 11, 282. [17] T. Hamasaki, Y. Sato, Y. Hatsuda, Agric. Biol. Chem. 1975, 39, 2337. [18] A. R. Howell, G. Pattenden, J. Chem. Soc., Perkin Trans. 1 1990, 10, 2715. [19] G. Vidari, A. Di Rosa, G. Zanoni, C. Bicchi, Tetrahedron: Asymmetry 1999, 10, 3547. [20] Z.-Y. Lu, H.-J. Zhu, P. Fu, Y. Wang, Z.-H. Zhang, H.-P. Lin, P.-P. Liu, Y.-B. Zhuang, K. Hong, W.-M. Zhu, J. Nat. Prod. 2010, 73, 911. [21] Y. M. Chung, C. K. Wei, D. W. Chuang, M. El-Shazly, C. T. Hsieh, T. Asai, Y. Oshima, T. J. Hsieh, T. L. Hwang, Y. C. Wu, F. R. Chang, Bioorg. Med. Chem. 2013, 21, 3866. [22] X.-R. Liu, F.-H. Song, L. Ma, C.-X. Chen, X. Xiao, B. Ren, X.-T. Liu, H.-Q. Dai, A. M. Piggott, Y. Av-Gay, L.-X. Zhang, R. J. Capon, Tetrahedron Lett. 2013, 54, 6081. [23] L. J. Fremlin, A. M. Piggott, E. Lacey, R. J. Capon, J. Nat. Prod. 2009, 72, 666. [24] T. Bunyapaiboonsri, S. Yoiprommarat, K. Intereya, K. Kocharin, Chem. Pharm. Bull. 2007, 55, 304. [25] D. Li, Y. Xu, C.-L. Shao, R.-Y. Yang, C.-J. Zheng, Y.-Y. Chen, X.-M. Fu, P.-Y. Qian, Z.-G. She, N. J. de Voogd, Mar. Drugs 2012, 10, 234. [26] C.-L. Shao, Z.-G. She, Z.-Y. Guo, H. Peng, X.-L. Cai, S.-N. Zhou, Y.-C. Gu, Y.-C. Lin, Magn. Reson. Chem. 2007, 45, 434. [27] D. G. I. Kingston, P. N. Chen, J. R. Vercellotti, Phytochemistry 1976, 15, 1037. [28] J. Magano, M. H. Chen, J. D. Clark, T. Nussbaumer, J. Org. Chem. 2006, 71, 7103. [29] M. S. Morales-Ros, J. EspiÇeira, P. Joseph-Nathan, Magn. Reson. Chem. 1987, 25, 377. [30] Y. Takenaka, N. Hamada, T. Tanahashi, Phytochemistry 2005, 66, 665. [31] J. A. Elix, A. J. Jones, L. Lajide, B. J. Coppins, P. W. James, Aust. J. Chem. 1984, 37, 2349. [32] G. T. Yogui, J. L. Sericano, R. C. Montone, Sci. Total Environ. 2011, 409, 3902. [33] X. Fu, F. J. Schmitz, M. Govindan, S. A. Abbas, K. M. Hanson, P. A. Horton, P. Crews, M. Laney, R. C. Schatzman, J. Nat. Prod. 1995, 58, 1384. [34] X. Fu, F. J. Schmitz, J. Nat. Prod. 1996, 59, 1102. [35] W. A. Kçnig, K. P. Pfaff, W. Loeffler, D. Schanz, H. Zhner, Liebigs Ann. Chem. 1978, 8, 1289. [36] K. Shibata, T. Kamikawa, N. Kaneda, M. Taniguchi, Chem. Lett. 1978, 7, 797. [37] J. Hargreaves, J. Park, E. L. Ghisalberti, K. Sivasithamparam, B. W. Skelton, A. H. White, J. Nat. Prod. 2002, 65, 7. [38] K. Scherlach, H. W. Nîtzmann, V. Schroeckh, H. M. Dahse, A. A. Brakhage, C. Hertweck, Angew. Chem., Int. Ed. 2011, 50, 9843. [39] M. Chen, C.-L. Shao, X.-M. Fu, R.-F. Xu, J.-J. Zheng, D.-L. Zhao, Z.-G. She, C.-Y. Wang, J. Nat. Prod. 2013, 76, 1229. [40] Y.-F. Huang, X.-X. Li, G.-D. Chen, H. Gao, L.-D. Guo, X.-S. Yao, Junwu Xuebao 2012, 31, 769. [41] G. H. Yang, K. Yun, V. N. Nenkep, H. D. Choi, J. S. Kang, B. W. Son, Chem. Biodiversity 2010, 7, 2766. [42] M. S. R. Nair, S. T. Carey, Mycologia 1979, 71, 1089. [43] T. Itabashi, K. Nozawa, S. Nakajima, K. Kawai, Chem. Pharm. Bull. 1993, 41, 2040. [44] M. Yamazaki, Y. Maebayashi, Chem. Pharm. Bull. 1982, 30, 514. [45] J. B. Spencer, P. M. Jordan, J. Chem. Soc., Chem. Commun. 1992, 8, 646. [46] R. A. Werner, A. Rossmann, C. Schwarz, A. Bacher, H.-L. Schmidt, W. Eisenreich, Phytochemistry 2004, 65, 2809. [47] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
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[48] L.-M. Sun, S.-J. Sun, A.-X. Cheng, X.-Z. Wu, Y. Zhang, H.-X. Lou, Antimicrob. Agents Chemother. 2009, 53, 1586. [49] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, ©. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09 (Revision B.01), Gaussian, Inc., Wallingford, CT, 2010. [50] H. Sun, J. Phys. Chem. B 1998, 102, 7338. [51] C. Diedrich, S. Grimme, J. Phys. Chem. A 2003, 107, 2524. [52] A. Klamt, V. Jonas, J. Chem. Phys. 1996, 105, 9972. [53] A. Klamt, J. Phys. Chem. 1995, 99, 2224. [54] P. J. Stephens, N. Harada, Chirality 2010, 22, 229. Received April 10, 2014
575
Identification and Biological Evaluation of Secondary Metabolites from the Endolichenic Fungus Aspergillus versicolor by Xiao-Bin Li a ), Yan-Hui Zhou a ), Rong-Xiu Zhu b ), Wen-Qiang Chang a ), Hui-Qing Yuan c ), Wei Gao d ), Lu-Lu Zhang d ), Zun-Tian Zhao d ), and Hong-Xiang Lou* a ) a
) Department of Natural Products Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Science, Shandong University, No. 44 West Wenhua Road, Jinan 250012, P. R. China (phone: þ 86-531-88382012; fax: þ 86-531-88382019; e-mail: [email protected]) b ) School of Chemistry and Chemical Engineering, Shandong University, No. 27 Shanda Nanlu, Jinan 250100, P. R. China c ) Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan 250012, P. R. China d ) College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Jinan 250014, P. R. China
A chemical investigation of the endolichenic fungus Aspergillus versicolor (125a), which was found in the lichen Lobaria quercizans, resulted in the isolation of four novel diphenyl ethers, named diorcinols F – H (1 – 3, resp.) and 3-methoxyviolaceol-II (4), eight new bisabolane sesquiterpenoids, named (¢)-(R)-cyclo-hydroxysydonic acid (5), (¢)-(7S,8R)-8-hydroxysydowic acid (6), (¢)-(7R,10S)-10hydroxysydowic acid (7), (¢)-(7R,10R)-iso-10-hydroxysydowic acid (8), (¢)-12-acetoxy-1-deoxysydonic acid (9), (¢)-12-acetoxysydonic acid (10), (¢)-12-hydroxysydonic acid (11), and (¢)-(R)-11-dehydrosydonic acid (12), two new tris(pyrogallol ethers), named sydowiols D (13) and E (14), and fifteen known compounds, 15 – 29. All of the structures were determined by spectroscopic analyses, and a number of them were further identified through chemical transformations and electronic circular dichroism (ECD) calculations. Preliminary bioassays of these isolates for the determination of their inhibitory activities against the fungus Candida albicans, and their cytotoxicities against the human cancer cell lines PC3, A549, A2780, MDA-MB-231, and HEPG2 were also evaluated.
Introduction. – Among fungi, those that live in close association with other organisms are assumed to be exceptional generators of biologically active compounds [1]. Lichens harbor abundant and diverse fungal communities. Recent studies have demonstrated that endolichenic fungi that live asymptomatically in the thalli of lichens are valuable sources of novel bioactive natural products, such as the antibacterial coniothiepinol A [2], a cyclic pentapeptide with synergistic antifungal activity [3], cytotoxic chromone derivatives [4], perylenequinones [5], ophiobolane sesterterpenes [6], diterpenoid geopyxin B [7], a demethoxyviridin that inhibits Ab42 aggregation [8], and antiviral heptaketides [9]. However, chemical investigations of this group of fungi are rather limited. During our search for new cytotoxic natural products from endolichenic fungi [5], the crude extract of Aspergillus versicolor (125a), which inhabits the lichen Lobaria quercizans, collected in the Laojun Mountain of Yunnan Province in China, exhibited significant cytotoxicity against the human osteosarcoma cancer cell line U2OS with an Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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IC50 value of 21.53 mg/ml. Fractionation of the AcOEt extract of its solid-substrate fermentation culture afforded 14 new compounds, including four diphenyl ethers, 1 – 4, eight bisabolane sesquiterpenoids, 5 – 12, and two pyrogallol ethers, 13 and 14, together with 15 known compounds, 15 – 29. Herein, we describe the isolation, and structure elucidation of the isolated compounds, as well as the evaluation of their antifungal and cytotoxic activities. Results and Discussion. – The AcOEt extract of A. versicolor afforded 29 compounds by using a combination of size-exclusion, normal-phase, and reversedphase chromatography. Structure Elucidation. Diorcinol F (1) was found to have the molecular formula C15H16O5 by a combination of HR-ESI-MS (m/z 277.1072 ([M þ H] þ ; calc. 277.1071)) and 13C-NMR data, accounting for eight degrees of unsaturation. The IR spectrum revealed the presence of OH functions (3355 cm ¢ 1) and aromatic moieties (1662 and 1589 cm ¢ 1). The 1H- and 13C-NMR data, together with the HSQC data (Table 1), exhibited signals of two tertiary Me group (d(H) 2.07, d(C) 20.9, Me¢C(5); d(H) 2.07, d(C) 20.5, Me¢C(5’)), one MeO group (d(H) 3.70, d(C) 60.2, MeO¢C(2)), four aromatic CH groups (d(H) 6.36, d(C) 111.5, CH(4); d(H) 5.97, d(C) 109.3, CH(6); d(H) 6.38, d(C) 111.9, CH(4’); d(H) 6.07, d(C) 110.9, CH(6’)), three exchangeable Hatoms (d(H) 9.16 (2 H); d(H) 8.32 (1 H)), and eight aromatic quaternary C-atoms. Based on these data, compound 1 was deduced to be a dimeric ether consisting of 3,4,5trihydroxytoluene units similar to violaceol I (18) [10], with the exception of the presence of MeO¢C(2) moiety in 1 instead of HO¢C(2) in 18. This conclusion was also supported by the HMBC from MeO¢C(2) to C(2) (d(C) 135.8) (Fig. 1). Accordingly, the structure of compound 1 was unambiguously determined as 3-(3-hydroxy-2methoxy-5-methylphenoxy)-5-methylbenzene-1,2-diol, which is structurally related to the fungal metabolites diorcinols B – E [11]. The trivial name diorcinol F was given to this new compound. Diorcinol G (2) had the molecular formula of C24H30O3 , deduced by the same strategy as for 1, indicating ten degrees of unsaturation. The presence of OH groups and aromatic moieties in 2 was inferred from its IR absorption bands at 3338, 1611, and 1598 cm ¢ 1. The 1D-NMR and HSQC data (Table 1) revealed the presence of six Me, two CH2 , and six aromatic/olefinic CH groups, ten aromatic/olefinic quaternary Catoms, four of which were oxygenated, and two exchangeable H-atoms. These data suggested that 2 was a derivative of diorcinol (16) [12] [13], with two additional prenyl ( ¼ 3-methylbut-2-en-1-yl) groups. One prenyl group at C(2) was determined by the 1 H,1H-COSY correlation H¢C(7) (d(H) 2.97)/H¢C(8) (d(H) 5.06), and the HMBCs from Me(10) (d(H) 1.54) to C(8) (d(C) 122.8) and C(9) (d(C) 130.2), from Me(11) (d(H) 1.48) to C(8) and C(9), and from H¢C(7) to C(1) (d(C) 154.1), C(2) (d(C) 118.6), and C(3) (d(C) 150.5) (Fig. 1). The other prenyl group at C(4) was identified by the 1H,1H-COSY correlation H¢C(12) (d(H) 2.98)/H¢C(13) (d(H) 4.88), and the HMBCs from resonance at Me(15) and Me(16) (d(H) 1.54) to C(13) (d(C) 122.8) and C(14) (d(C) 129.9), and from H¢C(12) to C(3), C(4) (d(C) 123.0), and C(5) (d(C) 135.3) (Fig. 1). Thus, the structure of compound 2 was as depicted. Diorcinol H (3) had the molecular formula C15H14O4 based on HR-ESI-MS (m/z 257.0818 ([M ¢ H] ¢ ; calc. 257.0808)) and 13C-NMR analyses, which revealed nine
a
6.07 (s) 2.07 (s) 2.07 (s) 3.70 (s)
6.38 (s)
5.97 (s)
6.36 (s)
1.54 (s) 1.54 (s)
1.54 (s) 1.48 (s) 2.98 (d, J ¼ 6.0) 4.88 (t, J ¼ 6.0)
2.97 (d, J ¼ 6.0) 5.06 (t, J ¼ 6.0)
5.97 (s) 2.14 (s) 2.12 (s)
6.17 (s)
5.88 (s)
6.54 (s)
d( H )
d(C )
d( H) 150.8 135.8 150.6 111.5 132.7 109.3 144.5 134.4 146.6 111.9 127.5 110.9 20.9 20.5 60.2
2
1 d(C )
23.1 122.8 130.2 25.5 17.5 25.4 122.8 129.9 25.5 17.6
154.1 118.6 150.5 123.0 135.3 114.1 159.2 99.1 158.3 109.2 139.5 106.2 19.2 21.3 2.69 (s) 2.73 (s) 3.87 (s)
6.56 (s)
6.80 (s)
6.60 (s)
d( H )
3 d(C ) 149.1 130.5 147.2 115.0 125.1 116.9 157.3 95.8 155.9 114.2 131.7 115.7 24.0 24.4 60.5 55.9
6.20 (s) 2.06 (s) 2.14 (s) 3.74 (s)
6.20 (s)
5.93 (s)
6.39 (s)
d(C ) 146.8 133.1 148.3 106.6 126.5 107.4 150.6 128.0 150.6 108.2 134.2 108.2 21.1 21.1
d( H)
4
) Recorded at 600 and 150 MHz, respectively. Assignments were based on 1H,1H-COSY, HMQC, and HMBC experiments.
1 2 3 4 5 6 1’ 2’ 3’ 4’ 5’ 6’ 5-Me 5’-Me 2-MeO 3-MeO 7 8 9 10 11 12 13 14 15 16
Position
1.56 (s) 1.56 (s)
3.10 (d, J ¼ 7.2) 5.12 (t, J ¼ 7.2)
6.12 (s) 2.13 (s) 2.15 (s)
6.25 (s)
6.02 (s)
6.15 (s)
6.45 (s)
d(H )
15
22.3 122.8 130.1 25.6 17.6
154.2 117.0 156.3 111.7 136.3 111.5 158.9 101.3 158.4 110.2 139.8 108.5 20.9 21.2
d(C )
Table 1. 1H- and 13C-NMR Data a ) (in (D6 )DMSO) of Compounds 1 – 4 and 15. d in ppm, J in Hz. Atom numbering as indicated in the Formulae.
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Fig. 1. 1H,1H-COSY (— —) and key HMB (H ! C) correlations of compounds 1, 2, 4, 5, 9, 13a, and 14a
degrees of unsaturation. Its IR spectrum revealed the presence of OH groups (3351 cm ¢ 1) and an aromatic system (1612 and 1584 cm ¢ 1). The NMR data of 3 (Table 1) were similar to those of the co-metabolite 3,7-dihydroxy-1,9-dimethyldibenzofuran (20) [14] with the noticeable difference of additional resonances (d(H) 3.87; d(C) 60.5) due to a MeO group at C(2). This finding was supported by the HMBC of MeO¢C(2) to C(2) (d(C) 130.5). Thus, the structure of 3, named diorcinol H, was determined as depicted. 3-Methoxyviolaceol-II (4) was assigned the molecular formula C15H16O5 , which is the same as that of 1, based on a combination of HR-ESI-MS (m/z 277.1072 ([M þ H] þ ; calc. 277.1071)) and 13C-NMR data. The 1H- and 13C-NMR data (Table 1), together with the 2D-NMR data (Fig. 1), of 4 were similar to those of violaceol-II (21) [10]. The HMBC of MeO¢C(3) (d(H) 3.74) with C(3) (d(C) 148.3) evidenced that compound 4 was the 3-O-Me derivative of 21, which is consistent with its molecular formula. (¢)-(R)-Cyclo-hydroxysydonic acid (5) was assigned the molecular formula C15H20O5 by a combination of HR-ESI-MS (m/z 303.1203 ([M þ Na] þ ; calc. 303.1203)) and 13C-NMR data, indicating six degrees of unsaturation. Its IR spectrum revealed absorption bands at 3390 (OH), 1687 (conjugated C¼O group), 1619 and 1605 (aromatic ring) cm ¢ 1. The 1H-NMR data displayed a typical ABX spin system
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assignable to a 1,3,4-trisubstitued benzene ring at d(H) 6.84 (d, J ¼ 7.8, H¢C(3)), 7.35 (s, H¢C(6)), and 7.61 (d, J ¼ 7.8, H¢C(4)). Its 1H- and 13C-NMR, and HSQC data (Table 2) revealed other resonances including those of three tertiary Me (d(H) 1.13, d(C) 29.6, Me(12) and Me(13); d(H) 1.65, d(C) 24.4, Me(14)), three CH2 (d(H) 1.47, d(C) 44.3, CH2(10); d(H) 1.62 – 1.56, d(C) 18.7, CH2(9); d(H) 1.97, d(C) 40.3, CH2(8)) groups, two sp3 oxygenated quaternary C-atoms (d(C) 69.8 (C(7), C(11)), one conjugated COOH (d(C) 167.1, C(15)) group, and six aromatic C-atoms. The 1H,1HCOSY correlations revealed the presence of the spin system CH2(8)¢CH2(9)¢CH2(10) (Fig. 1). Furthermore, the HMBCs (Fig. 1) from H¢C(12) or H¢C(13) (d(H) 1.13) to C(10), C(11), C(12), and C(13), from H¢C(9) to C(7), from H¢C(8) to C(2) (d(C) 121.8) and C(14), and from H¢C(14) to C(2) and C(8) confirmed the presence of an aliphatic side chain at C(2) of the benzene ring. Based on these data, compound 5 was determined as a bisabolane-type sesquiterpenoid similar to hydroxysydonic acid [15], with the exception of the absence of two H-atoms. At present, this compound could be assumed to have the structure of A or B (Fig. 2). However, further comparisons of the 13 C-NMR chemical shifts of C(7) and C(11) in 5 with those of C(1) and C(5) in 1,2dioxepanes confirmed the structure A [16]. The absolute configuration of compound 5 was determined by comparing the experimental and calculated ECD spectra predicted by time-dependent density-functional theory (TDDFT). The absolute configuration of C(7) was assigned as (R) based on the finding that the experimental and calculated ECD spectra of 5 were in good agreement (cf. Fig. 4, a). (¢)-(7S,8R)-8-Hydroxysydowic acid (6), (¢)-(7R,10S)-10-hydroxysydowic acid (7), and (¢)-(7R,10R)-iso-10-hydroxysydowic acid (8) were found to be isomers with the same molecular formula of C15H20O5 , based on a combination of their HR-ESI-MS and 13C-NMR spectra. Their similar 1D-NMR spectra (Table 2) exhibited signals for three Me and two CH2 groups, one CH¢O group, two sp3 quaternary O-bearing Catoms, a 1,3,4-trisubstitued benzene ring containing an oxygenated aromatic C-atom, and one conjugated COOH group. These data suggested that compounds 6 – 8 were hydroxy derivatives of sydowic acid (22) [17]. Compounds 6 and 7 were determined as 8-hydroxy- and 10-hydroxysydowic acids, respectively, by the downfield shifts in the resonances of C(8) (d(C) 69.1) in 6 and of C(10) (d(C) 70.7) in 7. The differences in
Fig. 2. Structures of A, B, and the fragments I, II, III, and IV of compounds 13a and 14a
1.13 (s)
11 12 29.6 24.4 167.1
69.8 29.6
44.3
9.35 (br. s)
1.15 (s) 1.64 (s)
28.0 23.4 170.1
2.04 – 2.00 (m, Ha ), 24.5 1.94 – 1.88 (m, Hb ) 1.94 – 1.88 (m, Ha ), 32.9 1.60 – 1.56 (m, Hb ) 75.4 1.38 (s) 30.4
4.19 (d, J ¼ 6.6)
7.55 (s)
7.22 (d, J ¼ 8.4) 7.55 (d, J ¼ 8.4)
156.6 136.0 125.5 121.4 129.8 119.4 81.3 69.1
d(C)
d(C )
9.11 (br. s)
1.00 (s) 1.55 (s)
1.33 (s)
25.0 31.1 170.7
77.9 26.4
156.9 136.7 7.15 (d, J ¼ 8.4) 124.5 7.57 (d, J ¼ 8.4) 121.7 129.9 7.56 (s) 119.3 77.9 2.30 (dt, J ¼ 14.0, 3.6, Ha ), 27.9 2.14 (td, J ¼ 14.0, 3.6, Hb ) 2.05 (tt, J ¼ 13.2, 3.6, Ha ), 24.1 1.86 – 1.82 (m, Hb ) 3.51 – 3.48 (m) 70.7
d( H )
7 c) d(C)
9.49 (s)
1.19 (s) 1.60 (s)
1.33 (s)
24.9 29.6 170.2
70.7 27.7
155.4 135.5 7.08 (d, J ¼ 7.8) 126.6 7.55 (d, J ¼ 7.8) 121.5 129.7 7.56 (s) 119.3 88.4 2.49 (dt, J ¼ 12.0, 8.4, Ha ), 38.8 2.24 – 2.19 (m, Hb ) 25.5 2.11 – 2.05 (m, Ha ), 1.92 – 1.86 (m, Hb ) 3.89 (t, J ¼ 7.2) 87.0
d( H)
8 c)
2.02 (s)
1.38 – 1.25 (m, Ha ), 1.21 – 1.14 (m, Hb ) 1.38 – 1.25 (m, Ha ), 1.11 – 1.04 (m, Hb ) 1.74 – 1.67 (m) 3.87 (dd, J ¼ 10.0, 5.4, Ha ), 3.79 (dd, J ¼ 10.0, 6.6, Hb ) 0.84 (d, J ¼ 6.6) 1.57 (s)
1.85 – 1.74 (m)
8.06 (d, J ¼ 6.6) 7.52 (d, J ¼ 6.6)
7.52 (d, J ¼ 6.6) 8.06 (d, J ¼ 6.6)
d( H )
9 c)
171.5 21.1
16.9 30.3 171.5
32.5 69.5
33.6
21.3
154.0 125.1 130.3 127.9 130.3 125.1 74.9 44.3
d(C )
a ) Recorded at 600 and 150 MHz, respectively. Assignments were based on 1H,1H-COSY, HMQC, HMBC and NOESY experiments. b ) Data were recorded in ( D6 )acetone. c ) Data were recorded in CDCl3 .
1.13 (s) 1.65 (s)
1.47 (t, J ¼ 7.8)
10
13 14 15 1-OH 12-AcO 12-AcO
1.62 – 1.56 (m)
9
18.7
152.5 121.8 6.84 (d, J ¼ 7.8) 108.4 7.61 (d, J ¼ 7.8) 125.6 148.7 7.35 (s) 109.7 69.8 1.97 (t, J ¼ 7.8) 40.3
d( H)
d(H )
d(C )
6 c)
5 b)
1 2 3 4 5 6 7 8
Position
Table 2. 1H- and 13C-NMR Data of Compounds 5 – 9 a ). d in ppm, J in Hz.
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the 13C chemical shifts in the heterocyclic rings of 7 and 8 were consistent with those of linalool oxides [18] [19]. The relative configurations of these three compounds were determined by their NOESY correlations (Fig. 3). The NOEs H¢C(8)/Hb¢C(10), Hb¢C(10)/Me(13), and Me(14)/Me(12) in 6 indicated that H¢C(8) and Me(14) were on different faces. The correlations H¢C(10)/Ha¢C(9), Ha¢C(9)/Ha¢C(8), Me(14)/ Hb¢C(8), and Hb¢C(8)/Hb¢C(9) in 7 evidenced that H¢C(10) and Me(14) were on different faces. Similarly, the NOEs Me(14)/Ha¢C(9) and Hb¢C(9)/H¢C(10) in 8 indicated that Me(14) and H¢C(10) were on different faces. The absolute configurations of compounds 6 – 8 were determined by comparing the experimental and calculated ECD spectra predicted by TDDFT (Fig. 4, b – 4, d). The experimental and calculated ECD spectra of 6 were in good agreement. The calculated ECD spectra of 7 and of 8 were nearly mirror images of their experimental ECD spectra, respectively. (¢)-12-Acetoxy-1-deoxysydonic acid (9) was found to have the molecular formula C17H24O5 based on its HR-ESI-MS (m/z 331.1520 ([M þ Na] þ ; calc. 331.1516)) and 13 C-NMR data, which revealed six degrees of unsaturation. The 1D-NMR data (Table 2) resembled those of 12-acetoxysydonic acid [20] with the exception of the A2B2 spin system (d(H) 7.52 (d, J ¼ 6.6, H¢C(2) and H¢C(6)) and 8.06 (d, J ¼ 6.6, H¢C(3) and H¢C(5))) in 9 rather than the ABX spin system in 12-acetoxysydonic acid, indicating the absence of a phenolic OH group in 9. The planar structure of 9 was confirmed by detailed analysis of its 2D-NMR data (Fig. 1).
Fig. 3. Selected NOESY correlations (H $ H) of compounds 6 – 8
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Fig. 4. Experimental and calculated ECD spectra of compound 5 (a), 6 (b), experimental ECD spectrum for 7 and calculated ECD spectrum for (7S,10R)-7 (c), and experimental ECD spectrum for compound 8 and calculated ECD spectrum for (7S,10S)-8 (d)
Compounds 10, 11, and 12 were found to be the 7-epimers of (þ)-(7S,11S)-12acetoxysydonic acid [20], (þ)-(7S,11S)-12-hydroxysydonic acid [21], and (þ)-(S)11-dehydrosydonic acid [20], respectively, based on comparisons of their NMR spectra, MS data, and optical rotation values with those in the literature. Because there was only one stereogenic C-atom, the absolute configuration at C(7) in 12 was assigned as (R) by comparing its negative optical rotation value ([a] 25 D ¼ ¢ 30.0 (c ¼ 0.1, MeOH)) with the positive optical rotation value of (þ)-(S)-11-dehydrosydonic acid ([a] 23 D ¼ þ 10.8 (c ¼ 0.11, MeOH)) [20]. From biogenetic considerations (Scheme), compounds 9 – 11 can be assumed to have the same absolute configuration, (7R), as that of 12. The molecular formulae of sydowiols D and E (13 and 14, resp.) were both determined as C21H20O7 based on their HR-ESI-MS data (m/z 383.1128 ([M ¢ H] ¢ ; calc. 383.1125)). Their 1H- and 13C-NMR spectroscopic data, which were similar to those of the co-metabolites sydowiols A and B (24 and 25, resp.) [22], indicated that both 13 and 14 consist of three ether-linked 3,4,5-trihydroxytoluene moieties. In our study, although compounds 13 and 14 could be separated by HPLC, these were
Scheme 1. Possible Biosynthetic Pathways to Compounds 1 – 15
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observed to be isomerized after standing in MeOH or MeCN. The methylation of a mixture 13/14 (MeI/K2CO3 ) afforded pentamethyl ethers 13a (C26H30O7 ) and 14a (C26H30O7 ), indicating the presence of five OH groups in 13 and 14. In the HMBC spectrum of 13a (Fig. 1), the correlations from H¢C(4) (d(H) 6.49) to C(2) (d(C) 137.8), attached to a MeO group (d(H) 3.89, d(C) 61.2), and C(3) (d(C) 153.4) attached to a MeO group (d(H) 3.87, d(C) 56.2), from Me¢C(5) (d(H) 2.25) to C(4) (d(C) 108.2), C(5) (d(C) 133.6), and C(6) (d(C) 112.6), and from H¢C(6) (d(H) 6.37) to O-bearing C(1) (d(C) 150.5) and C(2) indicated the partial structure I (Fig. 2). The HMBCs from H¢C(4’) (d(H) 6.08) to C(2’) (d(C) 137.9), attached to a MeO group (d(H) 3.99, d(C) 61.2), and O-bearing C(3’) (d(C) 152.6), from Me¢C(5’) (d(H) 2.07) to C(4’) (d(C) 110.1), C(5’) (d(C) 133.2), and C(6’) (d(C) 113.3), and from H¢C(6’) (d(H) 6.29) to oxygenated C(1’) (d(C) 150.3) and C(2’) indicated moiety II, whereas moiety III was suggested by the HMBCs from two H-atoms with signals at d(H) 6.46 (H¢C(4’’) and H¢C(6’’)) to C-atoms with signals at d(C) 153.1 (C(1’’) and C(3’’)), attached to MeO groups (d(H) 3.77, d(C) 56.3), and oxygenated C(2’’) (d(C) 130.3), and from Me(5’’) (d(H) 2.38) to C(4’’) (d(C) 106.3), C(5’’) (d(C) 135.5), and C(6’’) (d(C) 106.3), in addition to symmetry considerations. Due to the presence of a single MeO group, fragment II had obviously to be placed between fragments I and III. The structure of 14a was deduced by the same strategy that was used for the deduction of 13a. Three moieties, I, III, and IV (Fig. 2), were indicated by the detailed analysis of the HMBC data of 14a (Fig. 1), and III was connected to IV at C(1’) (d(C) 152.4) via an Oatom based on the HMBC between H¢C(6’) (d(H) 6.03) and C(2’’) (d(C) 130.3). Thus, 14a was unambiguously determined, and sydowiols D and E (13 and 14, resp.) were identified as shown. The assignments for the 1H- and 13C-NMR data of 13a and 14a are compiled in Table 3. Table 3. 1H- and 13C-NMR Data (in CDCl3 ) of Compounds 13a and 14a a ). d in ppm, J in Hz. Position
13a d( H )
1 2 3 4 5 6 2-MeO 3-MeO 5-Me 1’ 2’ 3’ 4’ 5’
6.49 (s) 6.37 (s) 3.89 (s) 3.87 (s) 2.25 (s)
6.08 (s)
14a d(C ) 150.5 137.8 153.4 108.2 133.6 112.6 61.2 56.2 21.7 150.3 137.9 152.6 110.1 133.2
d( H)
6.36 (s) 6.30 (s) 3.99 (s) 3.84 (s) 2.22 (s)
6.41 (s)
Position
13a d( H)
d(C )
d( H )
d(C )
6’ 2’-MeO 3’-MeO 5’-Me 1’’ 2’’ 3’’ 4’’ 5’’ 6’’ 1’’-MeO 3’’-MeO 5’’-Me
6.29 (s) 3.99 (s)
113.3 61.2
6.03 (s)
107.3
2.07 (s)
21.5 153.1 130.3 153.1 106.3 135.5 106.3 56.3 56.3 22.2
3.74 (s) 2.22 (s)
56.3 22.0 153.2 130.3 153.2 106.8 135.3 106.8 56.5 56.5 22.1
d(C ) 152.2 135.9 153.0 106.4 132.7 108.6 61.1 56.2 21.9 152.4 135.6 153.3 106.6 134.9
6.46 (s) 6.46 (s) 3.77 (s) 3.77 (s) 2.38 (s)
14a
6.41 (s) 6.41 (s) 3.69 (s) 3.69 (s) 2.33 (s)
a ) Recorded at 600 and 150 MHz, respectively. Assignments were based on HMQC and HMBC experiments.
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Diorcinol I (15) had the molecular formula of C19H22O3 , as established by a combination of HR-ESI-MS (m/z 299.1640 ([M þ H] þ ; calc. 299.1642)) and 1D-NMR data, indicating nine degrees of unsaturation. The UV, IR, and 1D-NMR data of 15 were similar to those of diorcinol G (2). Analyses of its 2D-NMR data revealed that the prenyl group at C(4) in 2 was absent in 15, and this finding was supported by the HMBCs from H¢C(4) (d(H) 6.45) to C(2) (d(C) 117.0), C(3) (d(C) 156.3), C(5) (d(C) 136.3), and C(6) (d(C) 111.5). This is the first report on the structure of 15, which was named diorcinol I. The known compounds were identified as diorcinol (16) [13], diorcinol D (17) [11], violaceol I (18) [23], cordyol C (19) [24], 3,7-dihydroxy-1,9-dimethyldibenzofuran (20) [14], violaceol II (21) [23], sydowic acid (22) [17], sydonic acid (23) [25], sydowiol A (24) [22], sydowiol B (25) [22], aversin (26) [26], 6,8-di-O-methylnidurufin (27) [27], 4hydroxybenzaldehyde (28) [28], and 3-formyl-1H-indole (29) [29] by comparing their spectroscopic data (MS, [a]D, UV, IR, and 1D-NMR) with those reported previously. Phenyl ether derivatives which turned out to be the major components in this study were previously obtained from cultured lichen mycobionts [10] [14] [30], lichens [31] [32], sponges [33] [34], and the fungi Aspergillus sp. [11 – 13] [21 – 23] [35 – 40], Penicillium sp. [20] [41], Hypocrea citrina [42], Emericella falconensis [43], Emericella violacea [44], and Cordyceps sp. [24]. To date, only one report appeared on the isolation of phenyl ethers from an endolichenic fungus [40]. It is noteworthy that phenyl ethers from natural lichens generally have carboxy groups, such as micareic acid, while phenyl ethers from the endolichenic fungus [40] and the cultured mycobionts [10] [14] [30] have no such group. In addition, this is the first report on the presence of bisabolane sesquiterpenes in an endolichenic fungus. Plausible biosynthetic routes to the new phenyl ethers and bisabolane sesquiterpenoids are outlined in the Scheme. The phenyl ethers are closely related to orsellinic acid and gallic acid, which are derived from the acetate-malonate pathway [45] and shikimic acid pathway [46], respectively. A series of dehydration, methylation, cyclization, oxidation reactions, and the addition of dimethylallyl diphosphate (DMAPP) unit to 16 lead to the formation of phenyl ethers 1 – 4 and 13 – 15. The bisabolane nucleus is built up via the mevalonic acid pathway [21]. Subsequently, 5 – 12 are formed through a series of oxidation, reduction, cyclization reactions, and esterification of the bisabolane nucleus. Biological Activities. All of the isolated compounds were evaluated for their cytotoxic activities against the PC3 (human prostate adenocarcinoma), A549 (human lung adenocarcinoma), A2780 (human ovarian carcinoma), MDA-MB-231 (human metastatic breast adenocarcinoma), and HEPG2 (human hepatocellular carcinoma) cancer cell lines by using the MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) assay [47]. As shown in Table 4, the cytotoxic activity displayed by A. versicolor (125a) was attributed to the presence of phenyl ethers. Among these, compounds 2 and 15 exhibited moderate cytotoxicities against all of the tested human cancer cell lines with IC50 values ranging from 19.0 to 36.3 mm. Compounds 3, 20, and 17 showed weak cytotoxicities against one to three of the tested human cancer cell lines. The results support the conclusion that the prenyl group (in 2 and 15) enhances cytotoxic activity, whereas an additional MeO group (in 3) reduces the activity. No cytotoxic activity was observed with the bisabolane sesquiterpenoids. However, it has
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Table 4. Cytotoxic and Antifungal Activities of Compounds a ) Compound
IC50 [mm] b ) c
3 2 15 20 17 18/21 19 27 Cisplatin Fluconazole
MIC (mg/ml) d
e
g
PC3 )
A549 )
A2780 )
MDA-MB231 f )
HEPG2 )
Candida albicans
> 50 24.0 0.5 24.7 1.4 39.4 1.7 31.6 0.8 > 50 > 50 31.9 2.3 18.0 1.5 NT
> 50 23.9 1.8 26.2 3.6 > 50 > 50 > 50 > 50 > 50 19.9 1.7 NT
> 50 31.0 0.6 36.3 2.1 > 50 > 50 > 50 > 50 33.7 2.5 14.9 2.3 NT
43.2 2.7 19.0 1.1 23.2 2.1 44.9 0.9 > 50 > 50 > 50 28.3 1.8 18.0 1.6 NT
> 50 24.3 0.9 27.3 1.7 45.2 1.2 29.8 1.0 > 50 > 50 > 50 14.3 1.5 NT
> 64 > 64 32 64 8 8 8 > 64 NT h ) 2
a
) Compounds which are not listed were inactive in all assays. Cisplatin and DMSO were used as the positive and negative controls, respectively, for the cytotoxicity assays; fluconazole and DMSO were used as the positive and negative controls, respectively, for the antifungal activity assay. b ) The IC50 values are means standard deviation of three independent replicates. c ) Human prostate adenocarcinoma. d ) Human lung adenocarcinoma. e ) Human ovarian carcinoma. f ) Human metastatic breast adenocarcinoma. g ) Human hepatocellular carcinoma. h ) NT, Not tested.
been reported that the condensation between phenolic bisabolane-type sesquiterpenoids and diphenyl ethers may increase their cytotoxicity [20]. The inhibitory activities of the isolated metabolites were also assessed against the fungus Candida albicans by using the microdilution method [48]. The known compounds 17, the mixture 18/21, and 19 exhibited significant activities with an MIC value of 8 mg/ml. Compounds 15 and 20 exhibited weak antifungal activities with MIC values of 32 and 64 mg/ml, respectively. All of the new compounds were inactive displaying MIC values greater than 64 mg/ml. Conclusions. – We reported 14 new compounds, including four diphenyl ethers, 1 – 4, eight bisabolane sesquiterpenoids, 5 – 12, and two pyrogallol ethers, 13 and 14, together with 15 known compounds, 15 – 29, isolated from the endolichenic fungus A. versicolor (125a). Phenyl ethers were confirmed as the major bioactive components in the extract of A. versicolor (125a) by evaluation of cytotoxic and antifungal activities. In addition, plausible biogenetic pathways of the new phenyl ethers and bisabolane sesquiterpenoids are also proposed. This work was supported by the National Natural Science Foundation of China (No. 81273383). We kindly acknowledge Mr. Shu-Qi Wang and Mr. Bin Ma for recording the NMR spectra, and Mrs. Yan-Hui Gao for the HR-ESI-MS data. Experimental Part General. TLC: Pre-coated SiO2 GF254 plates (Qingdao Marine Chemical Industry), visualization by using UV (254 nm) light or by spraying with 10% H2SO4/EtOH, followed by heating. Column chromatography (CC): silica gel (SiO2 , 200 – 300 mesh; Qingdao Marine Chemical Industry), octadecyl
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silica gel (ODS, 40 – 63 mm; Fuji Silysia Chemical, Ltd.), and Sephadex LH-20 gel (25 – 100 mm; Pharmacia Biotech). MPLC: Lisure EZ Purifier apparatus equipped with a UV/VIS dual-wavelength detector (210 and 254 nm; Lisure Science Corporation) and an ODS column (30 130 mm). Semi-prep. HPLC: Agilent 1100-G1310A isopump equipped with a G1322A degasser, a G1314A VWD detector (210 nm), and a ZORBAX SB-C18 column (9.4 250 mm, 5 mm). Optical rotations: Perkin-Elmer 241 MC polarimeter. UV Spectra: Shimadzu UV-2450 spectrophotometer; lmax (log e) in nm. CD Spectra: Chirascan spectropolarimeter; l (De) in nm. IR Spectra: Nicolet iN 10 Micro FTIR spectrometer; ˜n in cm ¢ 1. 1H- and 13C-NMR Spectra: Bruker Avance-DRX-600 spectrometer; at 600 (1H) and 150 MHz (13C); d in ppm rel. to Me4Si as internal standard, J in Hz; 2D spectra recorded with standard pulse programs and acquisition parameters. MS: API-4000 triple-stage quadrupole instrument for electrospray ionization (ESI) and Finnigan LC-QDECA mass spectrometer for HR-ESI; in m/z. Fungal Material. The endolichenic fungus A. versicolor (125a) was isolated from the lichen Lobaria quercizans, which was collected from Laojun Mountain, Yunnan Province, P. R. China. It was identified by Xin Zhang (Shandong Provincial Key Laboratory of Microbial Engineering, Qilu University of Technology, P. R. China) based on its nuclear ITS rDNA sequences (GenBank: GU227343). The strain, which was assigned the Accession No. 125a, was deposited with the lichen laboratory in the College of Life Sciences, Shandong Normal University, Jinan. The fungal strain was cultured in two 500-ml Erlenmeyer flasks, each of which contained 100 ml of potato dextrose broth (PDB), at 258 on a rotary shaker (110 rpm) for 8 d to prepare the seed culture. The fermentation was conducted in 20 Erlenmeyer flasks (500 ml), each of which contained 80 g of rice. Dist. H2O (120 ml) was added to each flask, and the contents were soaked overnight before autoclaving at 1208 for 30 min. After cooling to r.t., each flask was inoculated with 10 ml of the spore inoculum and incubated under static conditions at r.t. for 60 d. Extraction and Isolation. The culture medium containing the mycelium was cut into small pieces and extracted using AcOEt (3 8 l). The org. solvent was evaporated under reduced pressure to yield the crude extract (10.9 g), which exhibited cytotoxicity against the human U2OS cell line (IC50 50.35 mg/ml). The AcOEt extract was separated into ten fractions, Frs. A – J, on a SiO2 column (CH2Cl2/MeOH from 100 : 0 to 0 : 100). Fr. B (49.5 mg) was subjected to gel permeation chromatography (Sephadex LH-20; CH2Cl2/MeOH 1 : 1) to afford four subfractions, Frs. B1 – B4 . Fr. B3 (37.5 mg) was purified by HPLC (83% MeOH/H2O, 1.5 ml/min) to yield 2 (tR 46.0 min; 5.0 mg) and 15 (tR 18.0 min; 21.4 mg). Fr. D (1.3 g) was subjected to CC (Sephadex LH-20; same solvent system as described above) to afford four subfractions, Frs. D1 – D4 . Fr. D2 (213.2 mg) was purified by HPLC (71% MeOH/H2O; 1.8 ml/ min) to yield 26 (tR 38.0 min; 7.7 mg) and 27 (tR 41.0 min; 6.0 mg). Fr. D3 (944.0 mg) was separated by MPLC (ODS, MeOH/H2O from 50 : 50 to 100 : 0) to yield six subfractions, Frs. D3A – D3F . Fr. D3A (328.9 mg) was further purified by HPLC (60% MeOH/H2O; 1.8 ml/min) to afford 28 (tR 7.0 min; 23.5 mg), 29 (tR 9.0 min; 5.5 mg), 4 (tR 14.0 min; 5.2 mg), 1 (tR 18.0 min; 4.3 mg), and 3 (tR 26.3 min; 2.1 mg). Further separation of Fr. D3C (55.3 mg) by HPLC (71% MeOH/H2O, 1.8 ml/min) yielded 22 (tR 25.9 min; 18.2 mg). Fr. D3D (126.6 mg) was purified by HPLC (76% MeOH/H2O; 1.8 ml/min) to furnish 17 (tR 24.0 min; 90.7 mg). Compounds 20 (tR 13.8 min; 8.8 mg) and 16 (tR 17.3 min; 7.9 mg) were isolated from Fr. D4 (20.5 mg) by using HPLC (65% MeOH/H2O; 1.8 ml/min). Fr. F (4.5 g) was separated by CC (Sephadex LH-20; CH2Cl2/MeOH 1 : 1) to afford five subfractions, Frs. F1 – F5 . Of these, Fr. F3 (747.0 mg) was subjected to HPLC (60% aq. MeOH containing 0.2% CF3COOH) to yield 5 (tR 30.0 min; 0.5 mg), 9 (tR 27.0 min; 1.1 mg), 10 (tR 22.0 min; 67.1 mg), 11 (tR 24.0 min; 4.3 mg), and 12 (tR 37.0 min; 7.5 mg). The separation of the residue of Fr. F3 , which was performed using a procedure similar to that used for the separation of Fr. D3 , afforded 6 (HPLC; 28% MeCN/H2O (0.2% CF3COOH); 1.5 ml/min; tR 39.0 min, 1.1 mg), 7 (HPLC; 25% MeCN/H2O (0.2% CF3COOH); 1.8 ml/min; tR 24.0 min, 0.2 mg), 8 (HPLC; 28% MeCN/H2O (0.2% CF3COOH); 1.5 ml/ min; tR 31.0 min, 0.6 mg), and 23 (HPLC; 50% MeCN/H2O (0.2% CF3COOH); 1.8 ml/min; tR 20.5 min, 2.8 mg). Fr. F4 (3.4 g) was purified by MPLC (ODS; 50% MeOH/H2O) to yield four subfractions, Frs. F4A – F4D. Fr. F4A (2.7 g) was further purified by HPLC (47% MeOH/H2O; 1.8 ml/min) to yield 18 (tR 30.5 min; 652.8 mg) and 21 (tR 21.0 min; 572.2 mg) as the major compounds. Compounds 18 and 21 were separated as single compounds but isomerized to give a mixture after standing in MeOH, as reported in [10]. Fr. F4B (219.8 mg) was purified by HPLC (55% MeOH/H2O; 1.8 ml/min) to yield 19 (tR 21.0 min;
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24.3 mg), whereas the separation of Fr. F4C (162.4 mg) by HPLC (40% MeCN/H2O; 1.8 ml/min) afforded 13 (tR 23.6 min; 19.5 mg), 14 (tR 18.9 min; 14.5 mg), 24 (tR 27.8 min; 13.9 mg), and 25 (tR 21.4 min; 20.9 mg). Interconversions of 13 and 14, and of 24 and 25 were observed similarly to those of 18 and 21. Diorcinol F ( ¼ 3-( 3-Hydroxy-2-methoxy-5-methylphenoxy)-5-methylbenzene-1,2-diol; 1). Red amorphous solid. UV (MeOH): 278 (5.33), 227 (6.31). IR: 3355, 2933, 1662, 1589, 1510, 1459, 1342, 1320, 1229, 1193, 1138, 1076, 1022, 990, 826. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 277.1072 ([M þ H] þ , C15H17O þ5 ; calc. 277.1071). Diorcinol G ( ¼ 3-(3-Hydroxy-5-methylphenoxy)-5-methyl-2,6-bis(3-methylbut-2-en-1-yl)phenol; 2). Brown oil. UV (MeOH): 281 (3.65). IR: 3338, 2965, 2922, 2856, 1611, 1598, 1449, 1321, 1154, 1136, 1075, 1054, 1023, 991, 835. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 367.2267 ([M þ H] þ , C24H31O þ3 ; calc. 367.2268). Diorcinol H ( ¼ 4-Methoxy-1,9-dimethyldibenzo[b,d]furan-3,7-diol; 3). Brown amorphous solid. UV (MeOH): 231 (4.10). IR: 3351, 2971, 2927, 2851, 1612, 1584, 1519, 1450, 1407, 1383, 1269, 1241, 1200, 1149, 1135, 1079, 1000, 838. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 257.0818 ([M ¢ H] ¢ , C15H13O ¢4 ; calc. 257.0808). 3-Methoxyviolaceol-II ( ¼ 2-(2-Hydroxy-3-methoxy-5-methylphenoxy)-5-methylbenzene-1,3-diol; 4). Red amorphous solid. UV (MeOH): 278 (3.78), 228 (4.68). IR: 3278, 2940, 2861, 1604, 1515, 1463, 1427, 1361, 1319, 1206, 1143, 1083, 1063, 1022, 984, 820. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 277.1072 ([M þ H] þ , C15H17O þ5 ; calc. 277.1071). ( ¢ )-(R)-Cyclo-hydroxysydonic Acid ( ¼ (3R)-3-(4-Hydroxy-4-methylpentyl)-3-methyl-3H-1,2-benzodioxole-6-carboxylic Acid; 5). Colorless amorphous solid. [a] 25 D ¼ ¢ 40.0 (c ¼ 0.1, MeOH). UV (MeOH): 294 (3.83), 258 (3.85). CD (MeOH): 297 ( ¢ 1.83), 262 ( ¢ 1.81), 227 ( ¢ 2.30). IR: 3390, 3077, 2968, 1687, 1619, 1605, 1497, 1452, 1381, 1263, 1184, 1118, 1022, 941, 905, 829, 766. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 303.1203 ([M þ Na] þ , C15H20NaO þ5 ; calc. 303.1203). ( ¢ )-(7S,8R)-8-Hydroxysydowic Acid ( ¼ 3-Hydroxy-4-[(2S,3R)-3-hydroxy-2,6,6-trimethyltetrahydro-2H-pyran-2-yl]benzoic Acid; 6). Colorless amorphous solid. [a] 25 D ¼ ¢ 30.0 (c ¼ 0.1, MeOH). UV (MeOH): 281 (3.73), 228 (4.48). CD (MeOH): 298 ( þ 3.59), 250 ( ¢ 4.08). IR: 3242, 2972, 2937, 1699, 1572, 1511, 1438, 1370, 1261, 1220, 1130, 1103, 1065, 1046, 984, 956, 892, 771, 747. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 279.1232 ([M ¢ H] ¢ , C15H19O ¢5 ; calc. 279.1227). ( ¢ )-(7R,10S)-10-Hydroxysydowic Acid ( ¼ 3-Hydroxy-4-[(2R,5S)-5-hydroxy-2,6,6-trimethyltetrahydro-2H-pyran-2-yl]benzoic Acid; 7). Colorless amorphous solid. [a] 25 D ¼ ¢ 20.0 (c ¼ 0.1, MeOH). UV (MeOH): 278 (4.47), 228 (5.26). CD (MeCN): 255 ( þ 2.69), 240 ( þ 3.33), 225 ( þ 2.53), 218 ( ¢ 1.75). IR: 3263, 2972, 1694, 1573, 1511, 1436, 1378, 1267, 1221, 1180, 1108, 1068, 1038, 963, 833, 771, 749. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 279.1232 ([M ¢ H] ¢ , C15H19O ¢5 ; calc. 279.1227). ( ¢ )-(7R,10R)-Iso-10-hydroxysydowic Acid ( ¼ 3-Hydroxy-4-[(2R,5R)-5-(2-hydroxypropan-2-yl)-2methyltetrahydrofuran-2-yl]benzoic Acid; 8). Colorless amorphous solid. [a] 25 D ¼ ¢ 30.0 (c ¼ 0.1, MeOH). UV (MeOH): 283 (3.69), 228 (4.40). CD (MeCN): 297 ( ¢ 0.82), 248 ( ¢ 2.23). IR: 3241, 2974, 2930, 1694, 1574, 1510, 1417, 1375, 1289, 1212, 1182, 1095, 1062, 1022, 948, 892, 772, 749. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 303.1205 ([M þ Na] þ , C15H20NaO þ5 ; calc. 303.1203). ( ¢ )-12-Acetoxy-1-deoxysydonic Acid ( ¼ 4-[(2R,6S)-7-(Acetyloxy)-2-hydroxy-6-methylheptan-2yl]benzoic Acid; 9). Colorless oil. [a] 25 D ¼ ¢ 60.0 (c ¼ 0.1, MeOH). UV (MeOH): 227 (3.87). CD (MeCN): 245 ( ¢ 3.21), 233 ( ¢ 5.13). IR: 3436, 2941, 1715, 1611, 1461, 1409, 1392, 1373, 1249, 1183, 1124, 1036, 1019, 862, 780, 709. 1H- and 13C-NMR: Table 2. HR-ESI-MS: 331.1520 ([M þ Na] þ , C17H24NaO þ5 ; calc. 331.1516). Sydowiol D ( ¼ 3-[3-(2,6-Dihydroxy-4-methylphenoxy)-2-hydroxy-5-methylphenoxy]-5-methylbenzene-1,2-diol; 13)/Sydowiol E ( ¼ 3-[2-(2,6-Dihydroxy-4-methylphenoxy)-6-hydroxy-4-methylphenoxy]5-methylbenzene-1,2-diol; 14). Red amorphous solid. UV (MeOH): 277 (3.42). IR: 3388, 2923, 1703, 1598, 1509, 1461, 1312, 1189, 1065, 827. HR-ESI-MS: 383.1128 ([M ¢ H] ¢ , C21H19O ¢7 ; calc. 383.1125). Methylation of 13 and 14. MeI (0.2 ml) and K2CO3 (62.3 mg) were added to a mixture 13/14 (5.8 mg) in MeCN (7 ml), and the suspension was stirred at 408 for 50 h. At this time, TLC indicated the disappearance of 13 and 14. The mixture was cooled to r.t., and the reaction was quenched with 2m HCl soln. (22 ml). The mixture was diluted with H2O and extracted with CH2Cl2 (30 ml 3). The org. layers were combined, washed with H2O, saturated with NaCl, dried (MgSO4 ), filtered, and concentrated in
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vacuo. The residue was purified by HPLC (75% MeCN/H2O; 1.8 ml/min) to yield 13a (0.6 mg) and 14a (1.3 mg). 2,3,2’,1’’,3’’-Penta-O-methylsydowiol D ( ¼ 1-(2,3-Dimethoxy-5-methylphenoxy)-3-(2,6-dimethoxy-4methylphenoxy)-2-methoxy-5-methylbenzene; 13a). Colorless amorphous solid. UV (MeOH): 270 (3.53), 206 (5.26). IR: 2937, 2836, 1596, 1578, 1503, 1462, 1415, 1323, 1223, 1130, 1104, 1042, 1005, 967, 816, 775. 1H- and 13C-NMR: Table 3. HR-ESI-MS: 455.2068 ([M þ H] þ , C26H31O þ7 ; calc. 455.2064). 2,3,1’,1’’,3’’-Penta-O-methylsydowiol E ( ¼ 2-(2,3-Dimethoxy-5-methylphenoxy)-1-(2,6-dimethoxy-4methylphenoxy)-3-methoxy-5-methylbenzene; 14a). Colorless amorphous solid. UV (MeOH): 269 (3.01), 207 (4.92). IR: 2917, 2850, 1596, 1504, 1465, 1415, 1324, 1222, 1129, 1094, 1005, 811, 717. 1H- and 13 C-NMR: Table 3. HR-ESI-MS: 477.1879 ([M þ Na] þ , C26H30NaO þ7 ; calc. 477.1884). Diorcinol I ( ¼ 3-(3-Hydroxy-5-methylphenoxy)-5-methyl-2-(3-methylbut-2-en-1-yl)phenol; 15). Brown oil. UV (MeOH): 281 (3.30). IR: 3324, 2965, 2920, 2857, 1587, 1496, 1453, 1420, 1325, 1211, 1143, 1054, 1023, 989, 834. 1H- and 13C-NMR: Table 1. HR-ESI-MS: 299.1640 ([M þ H] þ , C19H23O þ3 ; calc. 299.1642). Biological Assays. Cytotoxicities of the compounds against the PC3, A549, A2780, MDA-MB-231, and HEPG2 cell lines were evaluated using the MTT assay [45], with cisplatin as positive control. Growth inhibitory activity against the fungus Candida albicans was assessed by the microdilution method [46], with fluconazole as positive control. Candida albicans was obtained from Affiliated Qianfoshan Hospital of Shandong University, P. R. China. Theory and Calculation Details. The calculations were performed with the Gaussian 09 program package [49]. Conformational searches were performed by MD simulations based on the COMPASS force field [50] and by scanning the potential energy surface (PES) on the main chain dihedral angles using the semiempirical AM1. All ground-state geometries were optimized at the B3LYP/6-31G* level at 298 K, followed by calculations of their harmonic frequency analysis to confirm these minima and then calculations of r.t. free energies. Electronic excitation energies and rotational strengths in gas phase and in MeOH/MeCN were calculated using TDDFT [51] at the same level in velocity formalism for the first 60 states. The solvent effect of MeOH/MeCN has been modeled by a conductor-like screening model for real solvents (COSMO) [52] [53]. The rotatory strengths were summed and energetically weighted following the Boltzmann statistics, and the ECD curves were simulated by using the Gaussian function [54]: De ðEÞ ¼
X 2 1 1 pffiffiffi DEi Ri e¢½ðE¢DEi Þ=s¤ ¢39 2:296 10 s p i
where s is half the band width at 1/e height, and DEi and Ri are the excitation energies and rotatory strengths for transition i, resp. A s value of 0.4 eV was applied for calculations.
REFERENCES [1] M. M. Wagenaar, D. M. Gibson, J. Clardy, Org. Lett. 2002, 4, 671. [2] Y.-C. Wang, S.-B. Niu, S.-C. Liu, L.-D. Guo, Y.-S. Che, Org. Lett. 2010, 12, 5081. [3] W. Wu, H.-Q. Dai, L. Bao, B. Ren, J.-C. Lu, Y.-M. Luo, L.-D. Guo, L.-X. Zhang, H.-W. Liu, J. Nat. Prod. 2011, 74, 1303. [4] F. Zhang, L. Li, S.-B. Niu, Y.-K. Si, L.-D. Guo, X.-J. Jiang, Y.-S. Che, J. Nat. Prod. 2012, 75, 230. [5] G. Li, H.-Y. Wang, R.-X. Zhu, L.-M. Sun, L.-N. Wang, M. Li, Y.-Y. Li, Y.-Q. Liu, Z.-T. Zhao, H.-X. Lou, J. Nat. Prod. 2012, 75, 142. [6] Q.-X. Wang, L. Bao, X.-L. Yang, D.-L. Liu, H. Guo, H.-Q. Dai, F.-H. Song, L.-X. Zhang, L.-D. Guo, S.-J. Li, H.-W. Liu, Fitoterapia 2013, 90, 220. [7] E. M. K. Wijeratne, B. P. Bashyal, M. X. Liu, D. D. Rocha, G. M. K. B. Gunaherath, J. M. UÏRen, M. K. Gunatilaka, A. E. Arnold, L. Whitesell, A. A. L. Gunatilaka, J. Nat. Prod. 2012, 75, 361. [8] Q.-C. Zheng, G.-D. Chen, M.-Z. Kong, G.-Q. Li, J.-Y. Cui, X.-X. Li, Z.-Y. Wu, L.-D. Guo, Y.-Z. Cen, Y.-Z. Zheng, H. Gao, Steroids 2013, 78, 896.
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[9] J.-W. He, G.-D. Chen, H. Gao, F. Yang, X.-X. Li, T. Peng, L.-D. Guo, X.-S. Yao, Fitoterapia 2012, 83, 1087. [10] Y. Takenaka, T. Tanahashi, N. Nagakura, N. Hamada, Chem. Pharm. Bull. 2003, 51, 794. [11] H.-Q. Gao, L.-N. Zhou, S.-X. Cai, G.-J. Zhang, T.-J. Zhu, Q.-Q. Gu, D.-H. Li, J. Antibiot. 2013, 66, 539. [12] J. A. Ballantine, C. H. Hassall, B. D. Jones, Phytochemistry 1968, 7, 1529. [13] A. N. Yurchenko, O. F. Smetanina, A. I. Kalinovsky, M. V. Pivkin, P. S. Dmitrenok, T. A. Kuznetsova, Russ. Chem. Bull., Int. Ed. 2010, 59, 852. [14] T. Tanahashi, Y. Takenaka, N. Nagakura, N. Hamada, Phytochemistry 2001, 58, 1129. [15] T. Hamasaki, K. Nagayama, Y. Hatsuda, Agric. Biol. Chem. 1978, 42, 37. [16] H. Kropf, H. Von Wallis, J. Chem. Res., Synop. 1983, 11, 282. [17] T. Hamasaki, Y. Sato, Y. Hatsuda, Agric. Biol. Chem. 1975, 39, 2337. [18] A. R. Howell, G. Pattenden, J. Chem. Soc., Perkin Trans. 1 1990, 10, 2715. [19] G. Vidari, A. Di Rosa, G. Zanoni, C. Bicchi, Tetrahedron: Asymmetry 1999, 10, 3547. [20] Z.-Y. Lu, H.-J. Zhu, P. Fu, Y. Wang, Z.-H. Zhang, H.-P. Lin, P.-P. Liu, Y.-B. Zhuang, K. Hong, W.-M. Zhu, J. Nat. Prod. 2010, 73, 911. [21] Y. M. Chung, C. K. Wei, D. W. Chuang, M. El-Shazly, C. T. Hsieh, T. Asai, Y. Oshima, T. J. Hsieh, T. L. Hwang, Y. C. Wu, F. R. Chang, Bioorg. Med. Chem. 2013, 21, 3866. [22] X.-R. Liu, F.-H. Song, L. Ma, C.-X. Chen, X. Xiao, B. Ren, X.-T. Liu, H.-Q. Dai, A. M. Piggott, Y. Av-Gay, L.-X. Zhang, R. J. Capon, Tetrahedron Lett. 2013, 54, 6081. [23] L. J. Fremlin, A. M. Piggott, E. Lacey, R. J. Capon, J. Nat. Prod. 2009, 72, 666. [24] T. Bunyapaiboonsri, S. Yoiprommarat, K. Intereya, K. Kocharin, Chem. Pharm. Bull. 2007, 55, 304. [25] D. Li, Y. Xu, C.-L. Shao, R.-Y. Yang, C.-J. Zheng, Y.-Y. Chen, X.-M. Fu, P.-Y. Qian, Z.-G. She, N. J. de Voogd, Mar. Drugs 2012, 10, 234. [26] C.-L. Shao, Z.-G. She, Z.-Y. Guo, H. Peng, X.-L. Cai, S.-N. Zhou, Y.-C. Gu, Y.-C. Lin, Magn. Reson. Chem. 2007, 45, 434. [27] D. G. I. Kingston, P. N. Chen, J. R. Vercellotti, Phytochemistry 1976, 15, 1037. [28] J. Magano, M. H. Chen, J. D. Clark, T. Nussbaumer, J. Org. Chem. 2006, 71, 7103. [29] M. S. Morales-Ros, J. EspiÇeira, P. Joseph-Nathan, Magn. Reson. Chem. 1987, 25, 377. [30] Y. Takenaka, N. Hamada, T. Tanahashi, Phytochemistry 2005, 66, 665. [31] J. A. Elix, A. J. Jones, L. Lajide, B. J. Coppins, P. W. James, Aust. J. Chem. 1984, 37, 2349. [32] G. T. Yogui, J. L. Sericano, R. C. Montone, Sci. Total Environ. 2011, 409, 3902. [33] X. Fu, F. J. Schmitz, M. Govindan, S. A. Abbas, K. M. Hanson, P. A. Horton, P. Crews, M. Laney, R. C. Schatzman, J. Nat. Prod. 1995, 58, 1384. [34] X. Fu, F. J. Schmitz, J. Nat. Prod. 1996, 59, 1102. [35] W. A. Kçnig, K. P. Pfaff, W. Loeffler, D. Schanz, H. Zhner, Liebigs Ann. Chem. 1978, 8, 1289. [36] K. Shibata, T. Kamikawa, N. Kaneda, M. Taniguchi, Chem. Lett. 1978, 7, 797. [37] J. Hargreaves, J. Park, E. L. Ghisalberti, K. Sivasithamparam, B. W. Skelton, A. H. White, J. Nat. Prod. 2002, 65, 7. [38] K. Scherlach, H. W. Nîtzmann, V. Schroeckh, H. M. Dahse, A. A. Brakhage, C. Hertweck, Angew. Chem., Int. Ed. 2011, 50, 9843. [39] M. Chen, C.-L. Shao, X.-M. Fu, R.-F. Xu, J.-J. Zheng, D.-L. Zhao, Z.-G. She, C.-Y. Wang, J. Nat. Prod. 2013, 76, 1229. [40] Y.-F. Huang, X.-X. Li, G.-D. Chen, H. Gao, L.-D. Guo, X.-S. Yao, Junwu Xuebao 2012, 31, 769. [41] G. H. Yang, K. Yun, V. N. Nenkep, H. D. Choi, J. S. Kang, B. W. Son, Chem. Biodiversity 2010, 7, 2766. [42] M. S. R. Nair, S. T. Carey, Mycologia 1979, 71, 1089. [43] T. Itabashi, K. Nozawa, S. Nakajima, K. Kawai, Chem. Pharm. Bull. 1993, 41, 2040. [44] M. Yamazaki, Y. Maebayashi, Chem. Pharm. Bull. 1982, 30, 514. [45] J. B. Spencer, P. M. Jordan, J. Chem. Soc., Chem. Commun. 1992, 8, 646. [46] R. A. Werner, A. Rossmann, C. Schwarz, A. Bacher, H.-L. Schmidt, W. Eisenreich, Phytochemistry 2004, 65, 2809. [47] T. Mosmann, J. Immunol. Methods 1983, 65, 55.
592
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
[48] L.-M. Sun, S.-J. Sun, A.-X. Cheng, X.-Z. Wu, Y. Zhang, H.-X. Lou, Antimicrob. Agents Chemother. 2009, 53, 1586. [49] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, ©. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09 (Revision B.01), Gaussian, Inc., Wallingford, CT, 2010. [50] H. Sun, J. Phys. Chem. B 1998, 102, 7338. [51] C. Diedrich, S. Grimme, J. Phys. Chem. A 2003, 107, 2524. [52] A. Klamt, V. Jonas, J. Chem. Phys. 1996, 105, 9972. [53] A. Klamt, J. Phys. Chem. 1995, 99, 2224. [54] P. J. Stephens, N. Harada, Chirality 2010, 22, 229. Received April 10, 2014
